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CRYPTOBIOTIC STAGES IN BIOLOGICAL SYSTEMS 



ELSEVIER MONOGRAPHS 
BIOSCIENCES SECTION 



Biological Subseries 




ELSEVIER PUBLISHING COMPANY 

AMSTERDAM-LONDON-NEW YORK-PRINCETON 



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PROCEEDINGS OF THE SYMPOSIUM 

ON 

CRYPTOBIOTIC STAGES IN 
BIOLOGICAL SYSTEMS 

5TH BIOLOGY CONFERENCE 'OHOLO' 1960 

SPONSORED BY 

THE ISRAEL INSTITUTE FOR BIOLOGICAL RESEARCH 

NESS-ZIONA (ISRAEL) 



Edited by 

NATHAN GROSSOWICZ 
Associate Professor of Bacteriology, Department of Bacteriology, 
Hebrew University-Hadassah Medical School, Jerusalem (Israel) 

SHLOMO HESTRIN 

Professor of Biochemistry, Department of Biochemistry, 
Hebrew University, Jerusalem (Israel) 

and 

ALEXANDER KEYNAN 
Scientific Director, Israel Institute for Biological Research, 

Ness-Ziona (Israel) 





ELSEVIER PUBLISHING COMPANY 

AMSTERDAM-LONDON-NEW YORK-PRINCETON 

1961 



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Library of Congress Catalog Card Number 61-14940 



With 37 illustrations and 31 tables 



ALL RIGHTS RESERVED. 
THIS BOOK OR ANY PART THEREOF MAY NOT BE REPRODUCED IN 
ANY FORM (INCLUDING PHOTOSTATIC OR MICROFILM FORM) 
WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS. 

PRINTED IN THE NETHERLANDS BY 
A. SIJTHOFF, THE HAGUE 



CONTENTS 

List of participants -/^^GIGXt^N: ^^^ 

Preface by the Editorial Committee • Aj^^^^^S^^'^^^s xi 

Opening address: / uj (^ — — M ^ . 

Cryptobiotic stages m biology . . V-sV •■ ■ ■ ■ •/•^i 
by H. O. Halvorson ! -^V ■^- • • J ^ I 1 

Cryptobiotic stages in bacteria: ^ ^ >^ 

Anhydrobiosis — a model of a cryptobiotic stage 

by A. KoHN AND M. Lion 15 

The bacterial endospore: A brief review of biochemical 

advances and some notes on sporogenesis 

by H. Orin Halvorson 32 

Studies on the germination of spores of B. Ucheniformis 

by A. Keynan and M. Halmann 64 

The biochemical nature of the dormant state in the 

bacterial endospore 

by H. Halvorson, R. O'Connor and E. Doi ... 71 

Cryptobiotic stages in fungi and parasitic worms: 

Hypobiosis in parasitic worms 

by G. WiTENBERG 97 

Hypobiotic phenomena in fungi and their significance 

in plant pathology 

by L Wahl 107 

Cryptobiotic stages in insects: 

Insect diapause in relation to the environment 

by A. D. Lees 120 

The endocrinology and biochemistry of insect diapause 

by A. D. Lees 132 

Environmental factors in interruption of development 

of Acrididae eggs 

by A. S. Shulov and M. P. Pener 144 



806 






VI CONTENTS 

Some observations on the role of diapause in the pheno- 
logy of insects in semi-arid zones 
by J. Harpaz 154 

Cryptobiotic stages in plants: 

Ecological problems of seed dormancy 

by D. KoLLER 159 

Role of plant hormones in dormancy of seeds 
by Alexandra Poljakoff-Mayber 165 

Dormancy and dormancy-breaking in seeds 
by S. Klein 175 

Biochemical changes in breaking and inducing dor- 
mancy in seeds 
by A. M. Mayer 191 

Rest in buds of woody plants 
by R. M. Samish 202 

Round table discussion: 

Common aspects of cryptobiosis in different biological 
systems 
Moderator: S. Hestrin 210 

Subject Index 227 



LIST OF PARTICIPANTS 

Y. Avi-DoR, Department of Biochemistry, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

A. Avivi, Department of Entomology, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

M. Bat-Miriam, Department of Entomology, Israel Institute 
for Biological Research, Ness-Ziona, Israel. 

Y. Bar-Sinai (Hirschberg), Section for Biotechnology, Israel 
Institute for Biological Research, Ness-Ziona, Israel. 

M. Bar-Zeev, Department of Entomology, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

A. Bekierkunst, Department of Bacteriology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

R. Ben-Gurion, Department of Bacteriology, Israel Institute 
for Biological Research, Ness-Ziona, Israel. 

N. Citri, Department of Bacteriology, Hebrew University — 
Hadassah Medical School, Jerusalem, Israel. 

R. Galun, Department of Entomology, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

R. Gavrielith-Shpan, Division of Plant Protection, Ministry 
of Agriculture, Jaffo, Israel. 

R. A. Goldwasser, Department of Virology, Israel Institute 
for Biological Research, Ness-Ziona, Israel. 

A. GoRDiN, Seed Research and Testing Division, Agricultural 
Research Station, Beith-Dagan, Israel. 

N. Grossowicz, Department of Bacteriology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

A. Halfon-Meiri, Seed Research and Testing Division, 
Agricultural Research Station, Beith-Dagan, Israel. 

Y. S. Halpern, Department of Bacteriology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

H. O. Halvorson, Department of Microbiology, School of 
Life Sciences, University of Ilhnois, Urbana, U.S.A. 

H. Halvorson, Department of Bacteriology, University of 
Wisconsin, Madison, Wise, U.S.A. 



VIII PARTICIPANTS 

I. Harpaz, Department of Entomology, Faculty of Agriculture, 

Hebrew University, Rehovot, Israel. 
Y. Henis, Department of Bacteriology, Faculty of Agriculture, 

Hebrew University, Rehovot, Israel. 
S. Hestrin, Department of Biochemistry, Hebrew University, 

Jerusalem, Israel. 
E. Kamon (Kaminski), Department of Zoology, Hebrew 

University, Jerusalem, Israel. 

E. Kaufmann, Department of Biochemistry, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

A. Keynan, Israel Institute for Biological Research, Ness- 
Ziona, Israel. 

S. H. KiNDLER, Department of Biochemistry, Israel Institute 
for Biological Research, Ness-Ziona, Israel. 

S. Klein, Department of Botany, Hebrew University, 
Jerusalem, Israel. 

A. M. Klingberg, Israel Institute for Biological Research, 
Ness-Ziona, Israel. 

A. KoHN, Department of Biophysics Israel Institute for Bio- 
logical Research, Ness-Ziona, Israel. 

D. KoLLER, Department of Botany, Hebrew University, 
Jerusalem, Israel. 

A. D. Lees, Agricultural Research Council, Unit of Insect 
Physiology, Cambridge, England. 

F. Mandelbaum, Department of Bacteriology, Hebrew 
University — Hadassah Medical School, Jerusalem, Israel. 

J. Margalit, Hebrew University, Jerusalem, Israel. 

A. M. Mayer, Department of Botany, Hebrew University, 
Jerusalem, Israel. 

R. MiLBAUER, Department of Bacteriology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

S. Nachmony, Department of Botany, Hebrew University, 
Jerusalem, Israel. 

D. Nachtigal, Weizmann Institute, Rehovot, Israel. 

E. Ohad, Ministry of Health, Jerusalem, Israel. 



PARTICIPANTS IX 

I. Ohad, Laboratory of Microbiological Chemistry, Hebrew 
University — Hadassah Medical School, Jerusalem, Israel. 

A. L. Olitzki, Department of Bacteriology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

Z. Olitzki, Department of Bacteriology, Hebrew University — 
Hadassah Medical School, Jerusalem, Israel. 

H. Oppenheimer, Department of Plant Physiology, Faculty of 
Agriculture, Hebrew University, Rehovot, Israel. 

R. Oren, Department of Biophysics, Israel Institute for Bio- 
logical Research, Ness-Ziona, Israel. 

M. P. Pener, Laboratory for Entomology and Venomous 
Animals, Department of Zoology, Hebrew University, 
Jerusalem, Israel. 

A. Poljakoff-Mayber, Department of Botany, Hebrew 
University, Jerusalem, Israel. 

L. Reinhold, Department of Botany, Hebrew University, 
Jerusalem, Israel. 

R. M. Samish, Division of Pomology and Viticulture, Agri- 
cultural Research Station, Faculty of Agriculture, Hebrew 
University, Rehovot, Israel. 

Z. Samish, Division of Food Technology, Agricultural Research 
Station, Rehovot, Israel. 

M. Shilo, Laboratory of Microbiological Chemistry, Hebrew 
University — Hadassah Medical School, Jerusalem, Israel. 

A. S. Shulov, Laboratory for Entomology and Venomous 
Animals, Department of Zoology, Hebrew University, 
Jerusalem, Israel. 

S. Talor, Hebrew University, Jerusalem, Israel. 

A. Traub, Department of Biochemistry, Israel Institute for 
Biological Research, Ness-Ziona, Israel. 

I. Wahl, Department of Plant Pathology, Faculty of Agri- 
culture, Hebrew University, Rehovot, Israel. 

G. WiTENBERG, Department of Parasitology, Hebrew Univer- 
sity — Hadassah Medical School, Jerusalem, Israel. 

B. WoLMAN, Ramat-Chen, Israel. 



X PARTICIPANTS 

S. Yathom, Department of Entomology, Agricultural Research 

Station, Rehovot, Israel. 
J. YosHPE, Laboratory of Microbiological Chemistry, Hebrew 

University — Hadassah Medical School, Jerusalem, Israel. 
R. Zelikson (Bentovim) Laboratory of Microbiological 

Chemistry, Hebrew University — Hadassah Medical School, 

Jerusalem, Israel. 
I. ZuKERMAN, Section for Biotechnology, Israel Institute for 

Biological Research, Ness-Ziona, Israel. 
J. Zydon, Israel Institute for Biological Research, Ness-Ziona, 

Israel. 



PREFACE 

Specialization in modern biology has assumed an extreme 
form. In this situation, even within a small country such as 
Israel, communication between speciaUsts could disappear 
entirely if it were not actively encouraged. At our annual 
'Oholo' conference, procedures which foster the desired inter- 
discipHnary communication in biology are being followed. The 
participants at these conferences represent widely divergent 
approaches to the biological method. They come from the 
different scientific institutions of Israel, and increasingly also 
from distant countries now that funds and facilities for travel 
have become more easily available. Subjects upon which dis- 
cussion at these meetings has been centered* belong to areas 
in which different biological disciphnes overlap and interlock. 
Ample allowances of time for formal and informal discussions, 
conducted against the serene background of an ancient and 
beautiful lake in Galilee, have complemented and greatly 
enhanced the value of the formal programs. 

The titles of the annual conferences convened as from the 
spring of 1956 at Oholo have designated a very wide area of 
discussion; yet this is, in fact, narrower than the one which 
we have covered. 

The subject of this year's meeting, 'Cryptobiotic Stages in 
Biological Systems', was one which has received less attention 
in the scientific literature than it probably deserves. With the 
notable exception of an important recent review on 'The Pro- 
blem of Anabiosis or Latent Life : History and Current Con- 
cept', by Prof. D. Keilin, the interdisciplinary approach in this 
area has been the exception rather than the rule. We are hoping 
that this book will help in some ways to answer the need. 



1956 Bacterial Genetics 

1957 Tissue Cultures in Virological Research 

1958 Inborn and Acquired Resistance to Infection in Animals 

1959 Experimental Approach to Mental Diseases 



XII PREFACE 

The scientific proceedings of the 'Oholo' conference are now 
being pubHshed for the first time in book form. The Editors 
gladly take this first opportunity to express their thanks to the 
participants in these conferences and to the organizing com- 
mittees for their generous gifts of time and effort which made 
this pubhcation possible. 

The Board of Editors hereby gratefully acknowledges the 
help of Mrs. B. Wolman in the preparation of the monograph 
and of Mr. L. Hirschberg-Bar-Sinai in the editing of the Round 
Table discussion. 



CRYPTOBIOTIC STAGES IN BIOLOGY 

H. ORIN HALVORSON 

School of Life Sciences, University of Illinois, Urbana, III. (U.S.A.) 

The term cryptobiosis was first introduced by Keilin^ in his 
Leeuwenhoek Lecture given at the University of Cambridge. 
He intended this term to include all forms of latent life, such as 
the anabiosis suggested by Preyer- and the abiosis introduced 
by Schmidt-*^. In his discussion of this pseudo-lifelessness, he 
treated the dormant state as though it were synonymous with 
cryptobiosis; but in his classification of the related terms he 
differentiated between them regarding the dormant state as 
encompassing hibernation, diapause, and quiescence, states 
where there is no growth but some metabohc activity. It would 
appear to me that, in many areas of biology, the term dormancy 
has been used to denote latent life, a state that shows metabolic 
activity only when a suitable environment arises. I can agree 
with Keilin that hibernation is not the same as cryptobiosis, in 
that in the former there is a positive metabolism even though it 
may be reduced to a low level, whereas in the latter no positive 
metabolism is perceptible. In this discussion, however, I shall 
use the term dormancy as it has been used in the past — by 
bacteriologists to describe bacterial spores and by zoologists to 
describe protozoan cysts. In these cases it is really analogous to 
cryptobiosis. As indicated in Keilin's outline, among various 
forms that exhibit reduced metabolism to a marked degree, 
there is no sharp dividing line between a state of 'hypometabo- 
lism' and 'ametabolism'. In dealing with the state of cryptobiosis. 
therefore, one must necessarily refer to some of the borderline 
cases. 

A state of cryptobiosis is certainly not limited to any single 
area of biology. One can find good examples among plants, 
animals and micro-organisms. Among these, one needs to 
mention: the seeds of plants; plant buds; the dehydrated forms 
of all kinds of micro-organisms, such as protozoa; bacteria, 

References p. 13 



2 H. O. HALVORSON 

fungi, and algae; viable forms rendered inactive by freezing; the 
cysts of protoza; and the spores of bacteria. I wish to discuss 
each of these in some detail under the headings of dehydrated 
forms, seeds, buds, cysts, insects, and spores. 

DEHYDRATED FORMS 

Keilin refers to this as a state of anhydrobiosis. In this state, 
forms seem to be lifeless because of lack of water. The inert, dry, 
yet viable protozoa were first observed by Leeuwenhoek when 
he showed that gutter-sand and dirt which had been kept in his 
laboratory for some time in a very dry condition, contained 
viable protozoa. They could be observed to be living and active 
immediately after sterile water was added to the material. 
Similar observations have been made by biologists from that 
day to the present. It is now well estabhshed, therefore, that 
many protozoa and some metazoans such as rotifers, tardi- 
grades, and nematodes, can be dried to the point where there is 
virtually no moisture left; these can be shown to be viable and 
unharmed, however, when rehydrated. Not only will they 
withstand this desiccation, but they can also be stored for long 
periods of time in the desiccated condition without, seemingly, 
much loss of viability. Similar experiments have been performed 
with many forms of fungi and bacteria. I am not familiar with 
the literature on algae, but it certainly must be true that many of 
their forms also can be desiccated in like manner and remain 
viable. This seems to be nature's way of preserving these forms 
through periods of drought and transport. 

It cannot be presumed, however, that complete desiccation 
and storage in the dry state does not damage the cells in some 
manner. This has been shown with bacteria that have been 
preserved by desiccation or lyophilization^' ^. When one 
makes quantitative studies of the number of viable cells present 
before and after drying and rehydration, one can show that 
many of the individual cells die in the process. Why some cells 
live and others die is certainly not known. It can be due to a 



CRYTOBIOTIC STAGES IN BIOLOGY 3 

difference in the biological state of the individual cells. It is also 
possible that the cells that die secrete substances which help to 
preserve and protect those that remain. It has been shown both 
with bacteria and protozoa that a few cycles of repeated drying 
and rehydration will kill all the cells if no opportunity is given 
for them to grow and repair damage between cycles. It is also 
clear from experiments that have been performed on bacteria, 
that damage can occur both during drying and rehydration as 
well as during the period of storage in the dry state^. This is 
clear from the varied quantitative results obtained with different 
methods of drying and rehydration. The degree of killing during 
drying is influenced greatly by the composition of the medium 
in which the cells are dried. An abundance of colloids will 
generally help to protect the cells; whereas metabolic end- 
products, such as acids and other small organic compounds will 
have an opposite effect, particularly as the concentrations are 
increased with the removal of water. The protective action of 
colloids, such as proteins, starches, and dextrins, may be due in 
part to a counteraction of the toxic effect of accumulated meta- 
bolic byproducts or to an antioxidant effect. The temperature of 
the material during drying can also have a marked effect. In 
general, the lower the temperature is during drying, the less the 
damage. It is for this reason in part, perhaps, that many cells 
can be preserved best when they are dried from the frozen 
state. Here, however, one does need to consider the additional 
damage that may occur during freezing. If cells are dried from 
the frozen state, it should be remembered that the rate of 
freezing is important as well as the nature of the menstruum that 
is frozen. 

Damage is also incurred during rehydration. With dehydrated 
bacteria and yeasts, the temperature and the rate of rehydration 
are important factors in controlling the extent of damage. 
Desiccated cells may sustain injury to their cell walls, so that 
during rehydration, soluble constituents leach out^. If the 
temperature and other environmental conditions are such that 
growth can be initiated at the outset, it may be possible for the 

References p. 13 



4 H. O. HALVORSON 

cells to repair this damage before the leaching and other sub- 
sequent undesirable changes become lethal. The manufacturers 
of bakers' yeast have been able to dry yeasts and retain a high 
degree of viability by carefully controlling the strain, growth 
conditions, method of desiccation, and method of rehydration. 
The problem is complex, and it is likely that optimum conditions 
will vary with each variety of cell. 

In bacterial cells, there seems to be some correlation between 
the size of the cells and the ease with which they can be preserved 
by desiccation. In general, the smaller cells can be preserved in 
this manner more successfully than the larger ones. It is easier, 
therefore, to keep cocci alive in a dry state than yeasts, and the 
vegetative cells of the Gram positive spore-forming bacteria are 
more easily damaged by drying than the smaller Gram positive 
lactic acid bacteria^. 

With most dried cells there is a slow 'die off' during storage. 
Here again, protective additives can have a marked effect on the 
stability of the cells. It is felt by many that the dam.age during 
storage may be due in part at least to oxidation and, therefore, 
that cells will be more stable if stored in an inert gas than in air 
or oxygen. This has been found to be true particularly with the 
viruses. Here, storage in the presence of air is quite damaging^. 
With these, to attain long time livability, it is necessary to dry 
and store the cells in an inert atmosphere at all times. Exposure 
to air even for a short period may be quite injurious, because, if 
the air comes in contact with the cells, enough oxygen is 
absorbed to produce products that are lethal. This lethal effect 
may be due to the formation of peroxides or hydroperoxides, 
which are known to be very toxic. 

Cells of protozoa, fungi, and bacteria that are properly dried 
can be kept viable for long periods of time. By this, we do not 
mean that all the cells in the population remain alive, but 
enough do, to allow one to recover an active culture when the 
material is rehydrated. This, of course, can happen even though 
only a very small fraction of the initial population remains 
viable. In our own laboratories, we have recovered active 



CRYPTOBIOTIC STAGES IN BIOLOGY 5 

cultures of pathogenic streptococci from lyophilized prepara- 
tions that have been stored under an inert gas at room tempera- 
ture for nineteen years. 

SEEDS 

It is by means of dormant seeds that nature provides a way of 
continuing the existence of many plant species. There are two 
types of these^o. One, a seed that remains dormant only as long 
as it remains dry. The process of germination is set in motion as 
soon as the seed comes in contact with moisture. The other type 
is a seed that remains dormant even though it has absorbed all 
the moisture that is needed. In this case, germination does not 
proceed until some external trigger mechanism sets the process 
in motion. 

The first represents a type of dehydrobiosis common to the 
seeds of cultivated plants, such as the gi*ains and maize. These 
seeds remain dormant during storage because they are kept dry. 
There may be a short period of after-ripening between the time 
they are harvested and the time they are planted, but in these 
seeds this period is short and occurs during the normal period of 
storage. When such seeds are planted, they imbibe moisture 
rapidly and germinate in a few days. The wild parents from 
which these cultivated plants have been derived probably did 
not have seeds of this kind, but through a long period of 
cultivation and natural selection, types have been developed 
which produce seeds that do not require any special trigger 
mechanism for germination. This is desirable in order that all 
the planted seeds germinate at the same time to produce plants 
that mature at about the same time. With seeds of this kind, 
there is some difference of opinion as to whether or not they are 
in a real state of cryptobiosis. In many studies on such seeds, it 
would appear that a slow rate of respiration goes on during the 
storage period even though the moisture content is low, and 
that this rate of respiration increases as the moisture content is 
increased^i. It is not certain, however, whether this respiratory 

References p. 13 



6 H. O. HALVORSON 

activity is from the seeds themselves or from a slow metabolism 
carried on by fungi, found on the seeds, which increase their 
rate of respiration when the moisture content is raised above the 
very minimum levels^"^. Certainly when such seeds are kept in 
environments where the moisture content is maintained below 
the critical levels of from 6 — 10% (depending upon the type 
and species), there is very little, if any, metabolism going on 
within the embryo of the seed itself. If this is not a true crypto- 
biotic state, it certainly is not far removed from one. 

The seeds of many weeds, flowering plants, vegetable plants, 
fruits, and legumes usually require special trigger mechanisms 
to germinate, in addition to the imbibition of water. In these, 
there may be a period of after-ripening or other special require- 
ments necessary. Some of these special requirements are the 
removal of inhibitors, the absorbtion of inducers, the breaking 
or damaging of hard seed coats, and the need for exposure to 
light to permit photoinduction. Whatever the mechanism — and 
it is often different for different species of seeds— many may 
remain dormant for many months or even years in an environ- 
ment where one would normally expect them to germinate^i. 
Those of you who have lived in the northern half of the United 
States of America, or in other areas that have similar climate, 
will know what I mean when I remind you of the difficulty of 
getting rid of crab grass in your lawn. Year after year one can 
remove new shoots in order to prevent the formation of any 
new seeds, and yet have the weed continue to come up year after 
year. Here a photoactivation is undoubtedly involved, but the 
biochemical mechanism that is triggered by this means is 
certainly not understood. In other cases, contact with oxygen 
through an exposure to normal atmosphere is necessary. Thus, 
many farmers have experienced the phenomenon of seeing new 
types of weeds appear in fields following deep plowing, weeds 
that had not been seen in those fields for many years. Here the 
deep plowing presumably brought deeply buried seeds to the 
surface where they could become exposed to oxygen and 
perhaps light, or both. 



CRYPTOBIOTIC STAGES IN BIOLOGY 7 

In many seeds there may be inhibitors of germination found 
in the fruit that covers the seed, or in the seed coating^^. As long 
as these are present, no germination can take place. During 
storage in the soil or other suitable environment, these inhibitors 
may leach out slowly or may even be destroyed by micro- 
organisms. Following their removal, germination can take 
place in a normal manner. In a single batch of seeds, not all of 
them will free themselves from the inhibitors at the same time. 
The different individuals may germinate at various times over 
a long period, thus helping to insure the propagation of the 
species. Germination in some seeds is triggered by a periodic 
change of temperature, that is, from storage at a low tempera- 
ture to storage at a high or moderate one. In some cases, more 
than one cycle of temperature change may be necessary. The 
mechanism involved here, as it must be in many of the other 
cases, must be biochemical, a temperature change being neces- 
sary for the proper sequence of biochemical changes. Un- 
fortunately, these phenomena are not understood, so that we do 
not have even fragmentary knowledge of the mechanism. 

Some seeds, like those of the legumes, have a very hard 
coating that is practically impervious to moisture and oxygen, 
so that the seed embryo within the shell is kept in a state of an- 
hydrobiosis, even though the environment outside the shell is 
conductive to germination^-*. These seed shells have interesting 
properties in that they will allow the moisture within the seed to 
escape, preserving the embryo through dehydration, but will 
not let water pass in the reverse direction. Such seeds can be 
made to germinate quickly by mechanically or chemically 
injuring the shell in order to let water in. In nature, such seeds 
may depend upon the action of bacteria from the outside to 
damage the coat, so that moisture can reach the embryo. 

BUDS 

The buds of plants usually remain dormant for a shorter or 
longer period, and many never open up. Whether or not they 
vegetate depends upon a number of conditions: such as favor- 

References p. 13 



8 H. O. HALVORSON 

able temperature, light, humidity; a nutrient supply; and 
certain conditions of equilibrium within the plant^-^. If a bud 
remains dormant for more than a year after its formation, it 
usually becomes latent, in which case it will open and grow 
only under rather unusual conditions'-^. 

Under normal conditions, buds will remain dormant only 
during the winter months. The exposure to the low temperatures 
of winter appears to be an essential feature in the breaking of 
the dormancy. When the warmer temperatures occur in the 
spring, the buds will begin to grow normally. The need for this 
exposure to the low temperatures of winter has been evidenced 
in some of the warmer climates where in certain years the 
temperature does not get low enough to properly condition 
these buds. Such buds, when they do begin to grow in the spring, 
are abnormal; and any flowers that are produced, are irregular. 
The cause of dormancy in buds is not well understood. Some 
believe it is due to a lowering of the oxygen level, but apparently 
this cannot be substantiated by the experimental evidence that 
is available. Considerable research has been directed toward 
breaking this dormancy by chemical means. For this purpose, 
ethylene, chlorohydrin, thiocyanates. thiourea, dinitrocresol, 
and dinitrophenol have been used, but there are many buds 
which do not respond to these chemicals. 

Latent buds may remain in the bark of the tree during the 
entire life of the plant and, as mentioned earlier, grow only 
under very unusual conditions. These latent buds are not loose 
in the bark, but continue to be attached and directly connected 
with the annual ring from which they originate. When a limb or 
stem of some size is cut, the numerous shoots that spring from 
near the cut edges of the bark come from these latent buds. 
These are som.etimes called adventitious buds. Practically 
nothing is known about the trigger mechanisms that initiate 
growth in these, but certainly this is nature's way of providing 
a mechanism for growth of the plant if the normal buds are 
destroyed. The most interesting examples of this that I have 
seen, are shoots that develop from these adventitious buds on 



CRYPTOBIOTIC STAGES IN BIOLOGY 9 

the burls of redwood trees when these are placed in a dish with 
moisture. 

The trigger mechanism involved here must certainly be bio- 
chemical, and an understanding of this would not only be very 
interesting but also very useful. 

CYSTS 

Among the protozoa, the cysts are perhaps the best example 
of a cryptobiotic state. In many respects these are similar to the 
seeds of plants and the spores of bacteria in that, while they 
remain as cysts, there is virtually no metaboHsm going on. In 
many cases these cysts remain as such only when they are stored 
at a low temperature or when they are dehydrated, but in other 
cases the cysts can remain stable for long periods of time in 
media where one would normally expect them to undergo 
excystment. With these, as in the case of some plant seeds, 
external factors other than moisture are needed to trigger the 
excystment process^'. Although a large number of environ- 
mental factors have been implicated by various workers, there 
is so much conflicting evidence that one cannot be certain which 
factors are important. In a few instances carefully controlled 
experiments tend to show that the principal factors are catalytic 
amounts of certain organic compounds, such as the potassium 
and sodium salts of 1-malic, citric, acetic, fumaric, oxalic, and 
lactic acids, with some carbohydrates acting as co-factors. These 
acids were even more effective when supplemented by low 
molecular weight organic compounds isolated from hay infusion. 
These unknown compounds were effective in concentrations as 
low as 10-^. In the absence of these acids and co-factors, the 
cysts can remain dormant for long periods in a medium that 
will support the growth of the normal adult and at temperatures 
that would favor this growth^^. In some instances these organic 
substances may be supplied by bacteria that are growing in the 
medium. 

In addition to the cysts, vegetative forms of protozoa of 

References p. 13 



10 H. O. HALVORSON 

various species may be put into a state of cryptobiosis by 
dehydration, as mentioned earlier, and also by a lowering of the 
temperature. Thus, many nonencysted forms of protozoa can 
be preserved for long periods of time by being suspended in a 
frozen medium. Here, there is a great deal of variation between 
species, some are sensitive to freezing whereas others are very 
resistant. Here, also, as with bacteria, the method of freezing, 
the ultimate temperature, and the constancy of the temperature 
at which they are stored can have an important bearing upon 
the longevity of the individual cells. 

INSECTS 

The eggs of insects, for example, the eggs of mosquitoes, 
have many properties in common with plant seeds and the cysts 
of protozoa. Under proper conditions these can be kept for 
long periods of time without loss of viability. Eventually they do 
die, which may indicate there is a low level of metaboHsm which 
uses up the reserve food. If stored at room temperatures, the 
life span is materially shortened. 

The hatching of these eggs may be compared to the germina- 
tion of seeds or the excystment of protozoan cysts. In most cases 
the hatching process needs to be triggered by some external 
factor, such as a favorable temperature, along with a lowering 
of the oxygen tension^^. In nature, these eggs may thus be 
preserved through the winter, but when spring comes and the 
temperature gets high enough for substantial growth of bacteria, 
the necessary lowering of O2 tension can occur through the 
metabolic activity of these organisms, and, as soon as the 
oxygen tension is brought to the proper low level, hatching will 
take place. In the laboratory the same thing can be accomplished 
by replacing the oxygen with nitrogen. Thus, persons who are 
working with mosquito eggs can store them for a long time 
without much loss of viability and hatch them at will. There are 
some mosquito eggs, however, that have a more complex trigger 
mechanism, in that they may require a change in temperature 



CRYPTOBIOTIC STAGES IN BIOLOGY 1 1 

from a low to a high value before they can be made to hatch^o. 
Hence, there are certain species of mosquitoes that may lay 
their eggs in the spring or summer, but the eggs will not hatch 
until they have been stored over the winter and then brought to 
the proper temperature in the spring-^. To the best of my 
knowledge, the biochemical changes that are involved are not 
known. 

In addition to the dormancy that is exhibited with these eggs 
of insects, it is also possible to get dormant forms in almost any 
of the developmental stages of the insects. These can be pre- 
served in a seemingly dormant state by a proper lowering of 
temperature. Many insects, therefore, will survive the frozen 
state. In general, however, there is a limit to how low the 
temperature can be brought. According to Prosser'^"^, at the 
University of Illinois, the temperature can be lowered until 
about 70% of the body water is frozen; below that, the organism 
dies. Depending upon the species, the temperature may be 
brought considerably, below the freezing point of water before 
a lethal temperature is reached. It is presumed that a consider- 
able amount of organic material is dissolved in the remaining 
water, so as to effectively lower the freezing point. In other 
words, the organisms have a built-in antifreeze system. At these 
low temperatures, the metabolism is reduced almost to, if not to, 
zero, so that these also may be regarded as being in a state of 
cryptobiosis. 

SPORES 

Among the bacteria, the spores are the best examples of a 
cryptobiotic state, although vegetative cells, as indicated above, 
can be brought into such a state through drying or freezing. 

The spores that are produced by some species of aerobes and 
anaerobes are the most resistant dormant cells known. Freshly 
produced and properly separated from their growth medium, 
they are truly inert and perhaps the best examples of a crypto- 
biotic state that can be found. Many studies have been made on 
the respiratory activity of such cells, but the data are conflicting. 

References p. 13 



12 H. O. HALVORSON 

Much of this is due to an improper understanding of the ease 
with which some of the cells can be made to germinate. Since 
the germinated cells do show considerable metabolic activity, 
one needs to exercise extreme care to insure that, in studies on 
spores, one does not deal with germinated cells-^. 

Before continuing this discussion we need to define our terms. 
The conversion of a spore to a mature vegetative cell involves 
two steps. The first, and one that can occur rapidly, involves 
only the activation of dormant enzyme systems that change the 
dormant cell to one that has an active metabolism. The second 
step involves the initiation of growth and finally the emergence 
of the new vegetative cell. Bacteriologists who are engaged in 
research on spores limit the term germination to the first step 
only. 

Several things occur simultaneously in this first change; the 
loss of refractility, the loss of heat resistance, and the gain of 
stainability. Recently we have found that the loss of resistance 
to octyl alcohol occurs, also. Germinated spores or vegetative 
cells are very sensitive to this chemical whereas spores are 
extremely resistant. During the initial change, many enzymes 
that are dormant in the spore become active and begin to 
function. This will include practically all the enzymes that are 
needed for energy for growth, such as those concerned in the 
electron transport system. Thus, clean spores will not show any 
oxygen uptake with glucose as a substrate, but germinated 
spores (using this term in the sense in which it was defined above) 
show a very rapid uptake of oxygen with that substrate. 

A number of agents can be used to trigger this germination 
process, such as a mixture of aiTiino acids and nucleotides, with 
or without previous heat shock, depending upon the species and 
past history of the spores. Some spores can be germinated with 
single amino acids, others with nucleotides alone, and some with 
various kinds of chelating agents. Regardless of the trigger 
mechanism used, dipicolinic acid, calcium ions, and some 
polypeptides are released from the spore simultaneously with 
the activation of the enzyme. Most workers believe that the 



CRYPTOBIOTIC STAGES IN BIOLOGY 1 3 

release of these substances is a necessary prerequisite for the 
activation of the enzymes. They believe that the enzymes are 
rendered dormant by incorporation into a complex. This 
complexing not only makes them inactive but also renders them 
resistant to heat and chemicals. 

In bacterial spores we find a number of phenomena that are 
common to other dormant systems. Aging simplifies the germi- 
nation requirements. This is comparable to the after-ripening 
process that goes on in certain seeds. Many fresh spores require 
heat shock in addition to the presence of the germination 
nutrients. Upon aging, the need for heat may disappear. Also, 
whereas fresh spores may require a number of substances to 
trigger the germination, after aging, some of the nutrients may be 
needed in reduced amounts or not at all. We beheve that these 
changes are due to autolytic enzymes which slowly release 
substances that can function as germination nutrients. In fact, 
we, as well as others, have shown that during aging, small 
amounts of alanine are released from the spore, and that this is 
one of the key substances needed for germination. 

In the study of bacterial spores, we are making progress 
toward an understanding of the mechanisms that are involved 
in conferring dormancy on cells. We are also learning how to 
break this dormancy. Further advances in this area may help us 
to better understand the mechanisms involved in dormancy in 
other areas of biology. 

REFERENCES 

1 D. Keilin, Proc. Roy. Soc. ( London), B, 150 (1958) 150. 

2 W. Preyer, Biol. Zentr., II (1891) 1. 

3 P. Schmidt, SSSR Academy of Science, Moscow, 1948. 

^ H. Proom, Symposium on Freezing and Drying, (R. J. C. Harris, Editor) 

Institute of Biology, London, 1951. 
5 M. Rhodes, J. Gen. Microbiol., 4 (1950) 450. 

** A. S. Lund, Ann. Kept. Hormel Inst. Univ. Minnesota, 1949-50, p. 47. 
' A. S. Lund, Ann. Kept. Hormel Inst. Univ. Minnesota, 1950-51, p. 70. 
^ J. A. Ulrich, Ann. Kept. Hormel Inst. Univ. Minnesota, 1947^8, p. 25. 
9 E. W. Flosdorf and S. Mudd, /. Immunol., 29 (1935) 389. 



14 H. O. HALVORSON 

1" E. H. Toole, S. B. Hendricks, H. A. Borthwick and V. K. Toole, 

Ann. Rev. Plant Physiol., 7 (1956) 299. 
1^ W. Crocker and L. V. Barton, Physiology of Seeds, Chronica Botanica 

Co., Waltham, 1957. 
1- M. Milmer and W. F. Geddes, Cereal Chem., 22 (1945) 484. 
13 M. Evenari, Botan. Rev., 15 (1949) 153. 
1^ E. O. C. Hyde, Ann. Botany (London), 18 (1954) 241. 
1^ J. F. Ferry and H. S. Ward, Fundamentals of Plant Physiology, The 

MacMillan Co., New York, 1959. 
1^ V. R. Gardner, Basic Horticulture, The MacMillan Co., New York, 

1959. 
1' W. J. VAN Wagtendonk, Biochemistry and Physiology of Protozoa, II; 

(S. H. Hunters and A. Lwoff, Editors) Academic Press, New York, 

1956, p. 87. 
1^ R. P. Hall, Protozoology, Prentice Hall, Inc., New York, 1959. 
1^ A. F. BoRG and W. R. Horsfall, Ann. Entomol. Soc. Am., 46 (1953) 472. 
•^0 W. H. Horsfall, Ann. Entomol. Soc. Am., 49 (1956) 66. 

21 I. N. McDaniel, Thesis, University of Illinois, 111., 1958. 

22 C. L. Prosser, personal communication. 

23 H. O. Halvorson, The Physiology of the Bacterial Spore, The Technical 
University Trondheim, Norway, 1958. 



ANHYDROBIOSIS — A MODEL OF A 
CRYPTOBIOTIC STAGE 

A. KOHN AND M. LION 

Department of Biophysics, Israel Institute for Biological Research, Ness-Ziona 

(Israel) 

The term anhydrobiosis, a state of ametabolic latent life due 
to dehydration was introduced by Giard in 1894^ as an addition 
to the terms osmobiosis, anoxybiosis, and cryobiosis. 

The process of lyophilization or freeze-drying discussed in 
this lecture is actually a combination of cryobiosis, anoxybiosis 
and anhydrobiosis, since the micro organisms are first frozen, 
then oxygen, air and water are removed by vacuum and a 
dehydrated viable product is obtained. 

The problems of anhydrobiosis in higher animals and plants 
(rotifers, nematodes, fungi, seeds of plants) are the subject of 
other lectures in this conference; some of them are treated by 
Keilin in his Leeuwenhoek Lecture-. In large animals, the 
maximal loss of water compatible with viability is about 92%, 
while in bacteria the loss of 99.5% or more of water is still 
compatible with continuing existence. Cryptobiosis in spore- 
forming bacteria is treated in a separate lecture. 

In the field of anhydrobiosis of vegetative bacteria, much 
information is available. In the National Type Culture Collection, 
out of 2,700 strains which were dried over phosphorus pentoxide, 
83 % remained viable after 14 years-^. Freeze-dried staphylococci, 
pneumococci, streptococci and some Gram negative bacteria 
were found to be viable after 17^ and 20 years^. Stamps showed 
that bacteria dried in a desiccator for 2-3 days under reduced 
pressure preserved their antigens and virulence for at least four 
years {S. typhi, P. pestis, P. leptoseptica, E. rhusipathiae, S. 
pyogenes, etc.). 

One of the latest papers on this subject has been published by 
Feldmann", who tested 100 strains of 58 species of bacteria. The 
bacteria were dried in vacuo, or freeze-dried in skim milk, and 

References p. 29 



16 A. KOHN AND M. LION 

aftei storage for 6 months and reconstitution were tested for 
viability, biochemical reactions, serological specificity, virulence, 
resistance to antibiotics, motility and pigment formation. Of 
all the strains tested, only three strains of pneumococci. one 
strain of meningococcus, one strain of TV. gonorhoeae and four 
strains of//, influenzae were lost. All the viable strains preserved 
their characteristics very well. 

Since the most efficient way of preserving micro-organisms in 
a desiccated state involves a freezing stage, we will briefly 
consider what happens to the micro-organisms when they are 
frozen. 

Experiments of Becquerel^ showed that bacteria withstand 
temperatures as low as 0.05" K. At very low temperatures ( — 190°- 
— 272°) all cell constituents are vitrified and dissociation and 
ionization are suppressed. At — 200° chemical reactions are 
eight miUion times slower than at 20°. The molecular state of 
some substances such as peroxides, catalase, hemoglobin and 
cytochrome changes; Keilin^, using this phenomenon of change 
in adsorption spectra in frozen material, found that the spores 
of B. subtiUs contained 6% of the cytochrome content of 
vegetative cells, a fact which he was not able to determine by 
measuring the respiratory activity of the spores. 

Practically, such low temperatures (below — 196°) are not 
employed for the preservation of viability in micro-organisms; 
rather they are frozen and kept at temperatures around — 80°. 

It has been shown by Weiser and Osterud^^ that death by 
freezing involves two processes, (a) a rapid or immediate death 
caused by freezing and thawing per se, and (b) a 'storage death' 
which is a function of time and temperature. Immediate death 
occurs at a stage of freezing when extracellular ice formation is 
being completed. As may be seen in Fig. 1, the rate of storage 
death is not uniform: it is more rapid at temperatures above 
— 30°, probably because the eutectic points of the solutes in 
media used for freezing are around this temperature. 

Death of cells due to freezing was attributed by Haines^^ to 
irreversible changes in the bacterial proteins, leading to their 



ANHYDRIOBIOSIS 



17 




-80 -100 -120 -140 -160 -180 

Temperature of storage 

Fig. 1 . Storage death of frozen cells of E. coli. 1 5-h culture of E. coli in 1 % 
peptone at pH 7.0, shell frozen in 0.1 -ml quantities. -1.5' and -5^ samples 
frozen at -15' then transferred to storage at required temperatures. Other 
samples frozen and stored at temperatures indicated. Storage time 3-5 h. 
(Modified from Weiser and Osterud^"). 



flocculation. He found that a protein extracted from P. pyocyanea 
coagulated when stored at — 2", but no such effect was observed 
when the protein was stored at — 20°. 

According to Straka and Stokes^- freezing produces several 
effects: it kills outright some of the cells, causes damage or 
reversible injury to others, while some cells entirely escape 
harm. The injury to bacteria may be detected by the ability of 
thawed cells to grow in a rich nutrient medium, but not in a 
minimal medium in which control, non-frozen bacteria grow 
well. At storage temperatures near 0"" a higher percentage of cells 
actually die than at — 30 \ when less bacteria are killed and 
more only injured: the injured ones can be saved on thawing by 
seeding them into a rich medium. The factor in the medium 
which was found to be responsible for the repair of the damage 
was trypticase (Baltimore Biological Laboratory), or rather 
some peptide in it. Amino acids and vitamins could not replace 
trypticase. 

Our own investigations of damage to the bacterial cell wall 

References p. 29 



18 



A. KOHN AND M. LION 



due to freezing of E. coli, as indicated by lysozyme sensitivity, 
showed that the cells are lysed by lysozyme immediately upon 
thawing to an extent many times greater than that of cells 
incubated at 37° for a short period of time after thawing (Fig, 
2)13. It could further be shown that following this immediate 



0.7 

0.6 



"T [ 1 1 1 1 T- 



in I I 



.-^0.5 

If) 

c 

0) 

-D 0.4 

"6 

u 

•.;3 0.3 

Q. 

o 

0.2 
0.1 



i^V. 



^^, 




\^ 



^ 



20 40 60 80 100 120 
Minutes after thawing 

Fig. 2. Loss of sensitivity to lysis by lysozyme of E. coli at different times 
after thawing. At times indicated by arrows 30 jug/ml of lysozyme was 
added. Dotted line indicates control lysis up to the time of addition of 
lysozyme. Each determination made in a separate tube from the same 
culture. Optical density measured at 540 mjn/(TQf.'^). 



recovery after thawing, considered to be osmotic or mechanical, 
there was a period of metabolic recovery which could be 
inhibited by starvation or metabolic poisons such as KCN, but 
not by chloramphenicol or 2,4-dinitrophenol (Fig. 3). 

Immediate damage to cells by freezing may be prevented by 
adding to the bacterial suspension 'protective colloids' such as 
skim milk. The explanation offered for the action of these 
colloids is that they prevent the death of bacteria by crushing 
due to extracellular ice when the inter-crystallic water films are 
thin. Colloids increase the thickness of these films and thus 



ANHYDRIOBIOSIS 



19 




10 20 30 40 50 60 
Minutes after thawing lysozime added 



Fig. 3. Sensitivity to lysis by lysozyme of frozen and thawed E. co/i, following 
starvation or KCN treatment. E. coli starved by aeration in phosphate 

buffer for 20 min before freezing (A A), or 0.005 M KCN added 

20 min before freezing (D D). Each point represents % lysis in 

20 min due to lysozyme added after thawing, at times indicated 2. 

Control: O O. 



leave enough space to accomodate the bacteria and prevent them 
from being crushed. We shall see later that these colloids have 
no action in protecting bacteria from death due to drying during 
the process of freeze-drying or after it. 

A very important chance discovery made first by Rostand in 
1946, and later, independently by Parkes^^. was that the addition 
of glycerol to animal cells (fowl sperm) protected them from the 
injurious and lethal effects of freezing. Bull semen has been 
successfully preserved in the frozen state for several years and 
calves have been produced by semen from a bull that had died 
three years previously. Thus a practical method was devised for 
freezing and storing semen, red blood cells, tissue culture cells 
and lately bone marrow cells. Recent information^^ indicates 
that bone marrow cells suspended in glycerol (10%) or frozen in 
glycerol are much more resistant to ionizing radiation than in 
the absence of glycerol. The effect of glycerol may be compared 
to the effect of drying since glycerol is a dehydrating agent. 

References p. 29 



20 A. KOHN AND M. LION 

Among the many processes of drying or dehydration of 
micro-organisms, freeze-drying has proved to be the least lethal 
and injurious. It is used nowadays not only for the storage of 
bacterial and viral strains but also for the preparation of living 
vaccines for prolonged storage, for preservation of some tissue 
used in transplantations, of plasma and of many labile biological 
materials such as enzymes, antibiotics, hormones, etc. 

In the process of freeze-drying of micro-organisms, a suspension 
of bacteria in a suitable medium is frozen (usually in a shell 
form on the walls of an ampoule) by dipping the ampoule into 
a carbon dioxide bath or into liquid air, and the water is then 
removed from the frozen material by sublimation in a vacuum 
apparatus. The rate of evaporation of water is such that the 
energy required for it is provided by the drying of the material, 
and thus the material remains frozen as long as any water is left 
in it. When the material is dry, the ampoule may be directly 
sealed in vacuo, or it may be filled before sealing with an 'inert' 
gas such as hydrogen or nitrogen. 

This process of lyophilization, — based on the discovery of 
Wollaston^^ in 1813 that water may be removed from ice by 
sublimation, — was first applied practically by ShackelU^ in 1909 
and later introduced for large scale preservation of biological 
materials by Flosdorf and Mudd^^' ^^. The optimal conditions 
in freeze-drying were extensively studied by Fry and Greaves^^ 
and Fry-^. An important contribution to the development of a 
practical suspension medium was made by Naylor and Smith^^, 
and their medium, composed of thiourea, ammonium chloride, 
sodium ascorbate and dextrin, is now widely used. 

Various workers have found empirically suspension media in 
which micro-organisms were well preserved in a dried state and 
remained viable after prolonged periods of storage. These media 
range from skim milk to serum and a mixture of serum and 
glucose and to Naylor's medium. 

Various theories have tried to explain why these particular 
substances protect bacteria undergoing drying, but none of them 
embraces all the materials involved or explains the results. 



ANHYDRIOBIOSIS 



21 



Recent investigations of Lion'-^ represent a great advance in 
the understanding of the process of freeze-drying and the 
importance of various factors involved. His results and their 
interpretation give a coherent picture of the process and present 
a reasonable hypothesis which not only accounts for most of the 
seemingly unrelated findings reported in the literature, but also 
allows the prediction of new lines of research and practical 
applications. 

The basic questions which Lion asked were: when and why 
do bacteria die during freeze-drying or during subsequent 
storage? What agent is lethal to micro-organisms during this 
process? 

To discover the lethal agent he first used 'naked' bacteria, 
i.e. bacteria suspended in distilled water, so as not to obscure 
the picture of possible protection by any of the chemical 
compounds present in the usual media. Most of the work was 



11 


«;^„ ^END PRIMARY 
-"^ — =« —-ik-r.._<^ DRYING 


.^___c_ 




\\ / " ' " " 
\ \ /BEGINNING 

\ \ C SECONDARY 

\ \ *^ DRYING 

\ a:. 


0) 
■*-> 
u 
o 
n 


\ 

\ 


-*A 


Log viable 

OD CD 


\ 

\ 

\ 

\ 
\ 
\ 

. 1 1 1 1 F 





5 6 

Hours 



Fig. 4. Kinetics of death of cells of E. coli during and following freeze- 
drying. Washed suspensions of E. coli about lO^Vml in distilled water. 
Between primary and secondary drying, various gases were introduced into 
ampoules: C = control in vacuum, H = hydrogen, A = air, O = oxygen. 



References p. 29 



22 



A. KOHN AND M. LION 



carried out on two strains of E. coli, which were osmotically 
stable enough to withstand suspension in distilled water; some 
experiments were done on other Gram negative bacteria. 

He found that the process of drying itself was quite innocuous 
to bacteria (even though suspended in water only), as long as 
they did not come into contact with air, or rather oxygen 
(Fig. 4). 

When an inert gas such as hydrogen or nitrogen was introduced 
into the dried material, the bacteria were preserved in a viable 
state as if sealed in vacuo. The lethal effect of air was proportional 
to its oxygen content, or to the partial pressure of the oxygen, 
and there was a reciprocal relation between the pressure of the 
air acting on the dried bacteria (in the range 50-760 mm Hg) 
and the time of exposure needed for inactivation. 







D 
t- 
0) 

X> -1 

D 
XI 

H- 
O 

-E -2 
n 

D 

> 

o 





^^ 


o r\ 


vV^ 


^^^^E 


\\ 


^^"^F 


Xh 


\ 


1 I 1 





12 3 4 

Hours of exposure to air 

Fig. 5. Protective properties of components of Naylors medium-'- on 
freeze-dried E. co// exposed to air. 1-ml suspensions of £". coli in the following 
media were exposed to air at the end of freeze-drying. Curve A = control 
in vacuum; B = full Naylor's medium; C = thiourea 1 %; D = thiourea 
0.5%; E = ammonium chloride 0.5%; F = sodium ascorbate 0.5%; 
G = distilled water; H = dextrin 2%. 



ANHYDRIOBIOSIS 23 

Lion then investigated the protective properties of Naylor's 
medium. Using each of its components separately and in com- 
bination, he found that only the thiourea protected bacteria 
from the lethal effects of oxygen (Fig. 5). 

Of various analogs of thiourea tested, such as methyl thiourea, 
dimethyl thiourea, trimethyl thiourea, thioacetamide, urethan, 
etc., only those in which the amino group was not totally 
substituted, and which had the S=C rather than the 0=C 
link, had protective powers. 

In view of reports on the protective properties of glucose, 
many sugars, monosaccharides, disaccharides, and trisaccharides 
were tested. In addition, the protective effects of inorganic salts 
with different cations and anions were tested, as well as those of 
reducing agents such as glutathione, cystein, cysteamine, sodium 
hydrosulfite, etc. 

Table I summarizes the results. It may be seen that of the 
inorganic salts, the Na cation, iodides, thiocyanates and nitrites 
are particularly effective in protecting bacteria against the lethal 
effect of air. The protective effect of Nal exceeds even that of 
thiourea. 

As to the sugars, their protective properties depended neither 
on their being fermented by the bacteria (lactose is fermented like 
glucose, but lacks protective properties), nor on their reducing 
character (m.altose which is reducing but not fermented lacks 
activity, while a-methyl glucoside which is not reducing and not 
fermented protects very well). 

Cystein and glutathione even increased the lethal effect of 
oxygen while colloids like albumin or dextran had no effect 
whatsoever. 

In another set of experiments it was found that the protective 
substances may be added as late as ten seconds before freezing, 
and still prevent death due to oxygen, thus indicating that they 
have no metabolic effect. They were, however, ineffective when 
added to the dried bacteria in the resuspending medium after 
exposure of the organisms to air. 

Another very important finding was that the degree of 

References p . 29 



24 



A. KOHN AND M. LION 



TABLE I 

PROTECTION OF FREEZE-DRIED CELLS OF E. CoU AGAINST LETHAL EFFECTS OF 

AIR (oxygen) by various SUBSTANCES 





* Protective 


* Prnterfivp 


Substance 




Substance 






properties 


properties 


Sugars 




Salts 




Glucose 


41 


NaCNS 


45 


Galactose 


31 


KCNS 


52 


Mannose 


42 






Fructose 


25 


MgCl2 


36 






MgS04 


0.5 


Lactose 


0.04 


N2HPO4 


O.OI 


Saccharose 


0.5 






Maltose 


0.2 










Water 


0.03 


Cellobiose 


1 


Ascorbate (0.5%) 


0.4 


Melibiose 


2 


Ammonium chloride (1 %) 


2.3 


Trehalose 


6 


Dextrin (2%) 


0.002 


a-Methyl glucoside 


32 


Thiourea (T.U.) (1%) 


42 


a-Methyl mannoside 


30 


Naylor's medium 


49 


Mannitol 


13 


Monomethyl T.U. 


59 


Inositol 


3 


Dimethyl T.U. (symmetr.) 


39 


Glucosamine 


42 


Dimethyl T.U. (asymmetr.) 


4.5 






Trimethyl T.U. 


5.5 






Urea(l%) 


7.8 


Salts 




Thioacetamide (1 %) 


4.7 


NaC10.16 M 


30 


Acetamide (1%) 


0.9 


KCl 


0.2 


Thiosemicarbazide (I %) 


0.9 


NaBr 


10 






KBr 


0.4 


Na2S204 (I %) 


10-3 


Nal 
KI 


80 

82 


Cysteamine (1%) 


10-2 


Cystein(0.75%) 


10-3 


Glutathione (0.16 M) 


10-5 


NaNOa 


64 






KNO3 (NH4CI) 


0.7 


Nutrient broth (2%) 


0.08 






Gelatin (1%) 


0.008 


LiCl 


12 


Skim milk 


0.03 


NaNOo 


44 






KNO2 


68 


Versene(0.1%) 


0.06 



* Per cent viability of freeze-dried cells of E. coli after 4 h contact with air. 



ANHYDRIOBIOSIS 25 

protection depended sharply on the proper ratio between the 
number of molecules of the protective substance and the number 
of bacteria. When the number of dried bacteria was increased 
tenfold, optimal viability was achieved when the quantity of the 
protecting substance was increased in the same ratio. In the 
case of Nal, approximately 100 million molecules of this 
compound per bacterial cell gives optimal protection against the 
lethal effects of air. 

An attempt was made to fit the heterogeneous data into a 
coherent hypothesis. It is clear that oxygen does not destroy the 
dried bacteria by oxidation or the formation of peroxides, since 
the protecting substances are mostly non-reductants. Moreover, 
some reducing substances with SH-groups, like glutathione, 
even enhance the lethal effect. 

Because of the electronic structure of the O2 molecule, we can 
assume that it is readily reactive with a number of metastable 
reactive metabolites. The latter may indeed be vital to the cell 
but ordinarily they are protected from direct attack by dissolved 
oxygen by the circumstance that oxygen has a low solubility and 
diffuses in water much more slowly than in a solid phase. 
Moreover, active metabolism of the cell makes the direct contact 
of the intermediates with oxygen unlikely. Little more can be 
said at this stage about the nature of these intermediates. 
However, it should be mentioned that Commoner detected free 
radicals in lyophilized biological materials-^. Also artificial free 
radicals in proteins produced by high doses of X-rays have been 
found to be stable in vacuo but to react quickly with oxygen and 
usually to disappear 2-^. It may be possible that these radicals are 
molecules in a triplet state as suggested by Szent-Gyorgyi-^. He 
claims that free energy in metabolic processes passes from place 
to place through substances in the reactive, triplet state {i.e. 
having electrons with 2 parallel spins). This state is metastable 
and its life span depends on the conditions prevailing. For 
instance, the presence of crystalline water or of hydration water 
around proteins makes it easier for the molecules to pass from 
singlet to triplet, i.e. to a more reactive state in which they 

References p. 29 



26 A. KOHN AND M. LION 

would react more easily with oxygen. In Szent-Gyorgyi's experi- 
ments, riboflavin phosphate which fluoresced in the presence of 
oxygen ceased to do so when certain substances, known as 
fluorescence quenchers, were added. These protective substances 
— KI, thiourea, cyanates, nitrites, etc.^ — which deactivate the 
reactive state sensitive to the magnetic field of oxygen are indeed 
the same that protect dried bacteria against the lethal effect 
of oxygen. 

Why are cations such as Na+ and Li+ protective, while K+ is 
not? Since the presence of crystalline water makes transition to 
the triplet state more probable, any ion which interferes with the 
formation of the crystal lattice should be protective, as indeed 
is the case with Na+. In the case of cystein and glutathione, 
which increase the lifetime of the metastable and reactive states, 
one would expect enhancement of the oxygen effect, as is 
actually observed. 

High mortality sometimes obtained on drying bacterial 
suspensions of low density in isotonic solutions may be obviated 
by adding more cells, alive or dead. Moreover, the same effect is 
produced by adding certain proteins, i.e. 'protective colloids' 

TABLE II 

PROTECTIVE PROPERTIES OF SODIUM IODIDE AND ALBUMIN ON 
LYOPHILIZED DRY CELLS OF E. CoU EXPOSED TO AIR'^ 



Cone. Concn. of Relative viability % 



Nal bacteria before No additives With 4% serum albumin 



/o 



drying A* B* A* B'^ 



1.75 


8.9- 


109 


0.02 


-0.002 






0.175 


1.5- 


1010 


60 


50 


47 


0.3 


1.75 


1.7- 


lO'i 


71 


69 


30 


11 


0.175 


1.9- 


10^1 


60 


0.7 







A* At the end of drying. 

B* After an exposure to air for 220 min at 28' 



ANHYDRIOBIOSIS 



27 



such as albumin (Table II). This finding also explains the con- 
flicting results obtained with various protective colloids and 
different bacteria when unequal concentrations of bacteria were 
used for drying. 

As has been mentioned previously, the 'protective colloids' 
perhaps diminish the number of deaths due to freezing alone : 
but if the optimal proportion between bacterial cells and their 
protective solution, for example Nal, has been established, the 
addition of the colloid may only make the situation worse. 

In conclusion I should like to mention some possible practical 
applications of these findings concerning the lethal effect of 
oxygen on dried bacteria. 

Analysis of the curve of inactivation of dried cells of E. coli 
by molecular oxygen or air (Fig. 6) indicated that the kilhng 
effect was not a monomolecular reaction and that it resembled 



100 




0.01 



2 3 4 5 

Hours of exposure to air 



Fig. 6. Kinetics of inactivation of freeze-dried E. coli exposed to air. 
Ampoules containing 1 ml of aqueous suspensions of E. coli, 
B/r (1.5 • lO^Vml) were exposed to air at the end of freeze-drying, for 
various periods of time. Curve A = experimental results; curve B = calcu- 
lated on the assumption that as regards its lethal effect the concentration of 
oxygen is reciprocal to the time of its action on dried bacteria (see text). 



References p. 29 



28 A. KOHN AND M. LION 

very much the curve of inactivation of the virus of poHomy- 
ehtis by formalin"^''. In both cases the lethal agent (oxygen or 
formalin) is in excess and its concentration does not change 
during the process of inactivation; in both cases the relative 
mortality of the viruses or bacteria is independent of their 
initial concentration. 

Card's empirical formula that may be fitted to the curve of 
inactivation is 

In — := a\n{\ + bt) (1) 

where yo is the viability of the virus at time 0, y the viability at 
time / and a and b are constants. For infinite time of reaction, a 
plot of log viability of cells against log time gives a straight line. 
If F is the concentration of formalin and C a constant, the 
kinetic equation derivable from the above would be of the form 

—dy 

= K.F.v 



dt 

Ko 

where K = — — and is a function of time. In the case of 

1 + Crt 

poliomyelitis, the change of the inactivation constant is ex- 
plained by a change in resistance of the virus to the action of 
formaldehyde in the course of the treatment, presumably 
because of change in the permeability of the virus membrane. 
As regards oxygen, one may postulate the existence in the 
bacterial cell of several sensitive targets (the metastable meta- 
bolic intermediates) that have different inactivation constants 
with regard to oxygen, the reaction of oxygen with a single 
molecule being of first order. The velocity of reaction between 
the cell and the oxygen would be proportional to the number of 
targets that have not yet reacted. The reaction constant, although 
fundamentally of first order, becomes dependent on time, as 
more and more targets (part of them vital) are knocked out. 



ANHYDRIOBIOSIS 29 

REFERENCES 

1 A. GiARD, Compt. rend. soc. hioL, 46 (1894) 497. 

■^ D. Keilin, Proc. Roy. Soc. London, 150 (1959) 149. 

3 M. Rhodes, /. Gen. Microbiol., 4 (1950) 450. 

■* R. Fasquelle and P. Barbier, Compt. rend. soc. bioL, 144 (1950) 1618. 

5 H. F. Swift, /. BacterioL, 33 (1937) 411. 

6 Stamp, J. Gen. Microbiol., / (1947) 251. 

7 S. Feldmann, Zentr. Bakteriol. Parasitenk., 176 (1959) 572. 

8 P. Bequerel, Compt. rend., 231 (1950) 1392. 

^ D. Keilin and E. F. Hartree, Antonie van Leemvenhoek. J. Microbiol. 
Seroi, 72(1947) 115. 

10 R. S. Weiser and C. M. Osterud, /. BacterioL, 50 (1945) 413. 

11 R. B. Haines, Proc. Roy. Soc. London, 124B (1938) 451. 

1- R. E. Straka and J. J. Stokes, J. Bacterial., 78 (1959) 181. 

13 A. KoHN, J. Bacterial., 79 (1960) 697. 

1^ A. S. Parkes, Sci. American, 194 (1956) 115. 

1^ M. Lion, personal communication, 1960. 

16 W. H. Wollaston, Phil. Trans. Roy. Soc. London, 70J (1813) 71. 

1" L. F. Shackell, Am. J. Physiol., 24 (1909) 325. 

1^ E. W. Flosdorf, Freeze-drying, Reinhold Publ. Co., New York, 1949. 

19 E. W. Flosdorf and S. Mudd, J. Immunol., 29 (1955) 389. 

20 R. M. Fry and R. 1. N. Greaves, J. Hyg., 49 (1951) 220. 

-1 R. M. Fry, in Biological Applications of Freezing and Drying, (Ed. R. J. C. 
Harris), Academic Press. Inc., New York, 1954, p. 215. 

-■2 H. B. Naylor and p. A. Smith, J. BacterioL, 52 (1946) 565. 

-3 M. Lion, Thesis, Hebrew University, Jerusalem, 1959. 

2^ B. Commoner, Conf. on Radio and Microwaves Spectroscopy, Duke Uni- 
versity Press, Durham, 1957. 

25 W. Gordy, W. B. Ard and H. Shields, Proc. Natl. Acad. Sci. U. S., 41 
(1955) 983. 

26 A. Szent-Gyorgyi, Bioenergetics, Academic Press. Inc., New York, 1956. 
" S. Gard, Ann. N. Y. Acad. Sci., 83 (1960) 638. 



DISCUSSION 

Avi-Dor: Some observations made in our laboratory by 
Mrs. Miller might be of interest here. When E. coli cells were 
suspended in distilled water, they swelled and lost potassium 
without losing sodium ions at the same rate. On addition of an 
oxidizable substance like glutamate no respiration occurred in 
these cells until potassium was also added. Addition of sodium 



30 A. KOHN AND M. LION 

did not have the same effect. Potassium iodide was more 
effective than several other potassium saUs. The potassium 
effect depended on the concentration of bacteria. 

Kohn: Dr. Avi-Dor mentioned the metabohc importance of 
potassium and sodium, and that this effect required a certain 
time to manifest itseff. In our laboratory Dr. Lion has per- 
formed a number of experiments in which addition of salt was 
made one second before freezing. Nevertheless the protective 
effect was obtained. Regarding the ion effect reported by you, 
is it a minimum effect or do you get an optimum curve? With 
glucose the viability rises with concentration until a certain 
maximum viability is reached. Further additions of glucose do 
not alter the viability. With thiourea and sodium iodide, how- 
ever, once you have reached the optimal concentration, further 
addition decreases the viability rapidly. 

Avi-Dor: Regarding your first question: we measure respira- 
tion which is a secondary effect of potassium. We do not know 
whether the primary effect is instantaneous or not. As to your 
second question, we did not reach an inhibitory concentration 
with potassium. 

Mayer: Have you tried to correlate the fluorescence quench- 
ing action of thiourea derivatives and their protective action? 
What is your evidence that free radicals are involved? Does an 
increase in thiourea concentration protect against a higher 
oxygen concentration? Finally has dinitrophenol any effect? 

Kohn: Only three thiourea derivatives were found to be 
active: thiourea, monomethyl thiourea and symmetric dimethyl 
thiourea. Regarding the relation between oxygen concentration 
and that of thiourea, no specific experiments were carried out. 

Shilo: In some bacteria, some unicellular algae and certain 
higher plants carotenoid pigments protect against photo- 
oxidative death. It occurs to me that a comparison between 
bacteria containing carotenoid pigments and mutants lacking 



ANHYDRIOBIOSIS 3 1 

carotenoid would be useful as a means of clarifying the mecha- 
nism of the oxygen effect. 

Hestrin : Is the oxygen-death a temperature-dependent reac- 
tion? By the way, if the specific target is inside the cell and the 
salt in question is outside, how does the interaction come about 
in the solid state? 

Kohn: In regard to your first question: when the dried 
ampoule is opened at — 80' and kept at that temperature the 
lethal effect does not occur. As to your second question, there 
could be a steric effect involving crystalline water and protein. 
The substrate acted upon may be a part of the electron transport 
system of the cell. 



THE BACTERIAL ENDOSPORE 

A BRIEF REVIEW OF BIOCHEMICAL ADVANCES AND 
SOME NOTES ON SPOROGENESIS 

H. ORIN HALVORSON 

School of Life Sciences, University of Illinois, Urbana, III. (U.S.A.) 

Three crucial observations have, in my opinion, stimulated 
many of the recent researches on spores. One, the observation 
by Hills^ that spores will germinate rapidly in the presence 
of a mixture of amino acids and nucleosides; two, the discovery 
in our laboratory that spores contain active heat-resistant 
enzymes-"^ and three, the observations made by Powell-^, that 
dipicolinic acid is a major constituent of spores. I want to take 
some time to discuss each of these observations. 

From early studies it was known that spores suspended in a 
growth medium would lose their heat resistance in a relatively 
short time. In looking into this further, Hills^ found that yeast 
extract had the same effect. He then proceeded to fractionate 
this to determine what nutrients were responsible for this effect, 
and discovered that spores would germinate in a few minutes in 
the presence of a few amino acids, with or without nucleosides 
and glucose. L-Alanine and adenosine were found to be sufficient 
for some species. This was studied in more detail by the late 
Joan Powell^ et a/. They found that a large percentage of the 
spores in a suspension (in the presence of the proper nutrilites) 
would simultaneously lose their heat resistance and refractility, 
and at the same time would become stainable. Many investiga- 
tors have confirmed these findings since they were made early in 
1950, In addition, the minimal germination requirements for 
other species have been determined. In most cases germination 
can be initiated by a few amino acids along with nucleosides, 
although some species will germinate with glucose only. 

As a result of the studies on the germination requirements, we 
have also gained a fairly good insight into the effect of environ- 



THE BACTERIAL ENDOSPORE 33 

mental factors upon germination. It is necessary, at this point, 
to indicate that germination is used here in a somewhat different 
sense than it has commonly been employed in the past. In older 
literature the word has been used to describe all of the changes 
taking place during the development from spore to fully grown 
vegetative cell. The changes observed by Hills and later by 
Powell and other workers occur very early in the process, and 
bacteriologists have come to use the term to encompass these 
early changes only, and to use the word outgrowth to represent 
all the other changes. 

A number of investigators have shown that many species of 
spores require a heat shock as a sensitizing mechanism before 
they will respond to germination nutrients. The length of the 
time of heating and the temperature required vary with different 
species, the age of the spores, and the conditions of storage. 
Freshly grown spores of Bacillus cereus can be adequately 
sensitized by heating to 65' for about 15 min. On the other hand, 
spores of Bacillus stearothermophilus have to be heated to 100° 
or more for an equivalent length of time". Most workers in the 
field believe that this heat sensitization initiates biochemical 
reactions that either release compounds to stimulate germina- 
tion or alter the permeability of the spore wall. In any event, 
precise information as to what actually does happen is not 
available. 

The rate of germination is notably influenced by the tempera- 
ture. Most of the investigators have been observing germination 
at room temperature, and it has been their common observation 
that it will not take place in a refrigerator or at a lower tempera- 
ture, nor will it occur at 60''^. On the other hand, Wynne^ 
claimed that spores from some species of anaerobes germinated 
at 75° when they were suspended in a solution containing 
glucose that had been autoclaved in an alkaline medium. 
Recently, Foster^ reported that Bacillus megatherium spores 
will germinate at temperatures between 70' and 100 . In our 
own laboratories, we find spores of B. cereus will germinate 
rapidly in the presence of L-alanine and adenosine at room 

References p. 59 



34 H. O. HALVORSON 

temperature but will not do so in the same menstruum if kept 
at 65°. 

The germination requirements are also affected by aging. 
Freshly produced and thoroughly cleaned spores have the most 
rigid requirements, but as they are aged the requirements 
generally become less so. The rate at which these changes take 
place depends upon the temperature of storage. Spores stored 
in a frozen state can remain unchanged for a very long period, 
whereas those stored in a refrigerator will change more rapidly. 
There is a marked difference in different species as to the rate at 
which these changes can take place. One of the most noticeable 
effects of aging is the disappearance of the need for heat sensi- 
tization. The changes that take place during aging are probably 
the same type as those which occur during heat shock. The only 
difference is one of time. In our laboratories, we have detected 
free L-alanine in the same supernatant liquor in which spores 
have been stored and aged, but this amino acid cannot be found 
in the supernatant liquor from freshly prepared clean spores^^. 
Alterations probably occur within the spore during aging, 
resuhing in the release of chemicals required for germination. 
It is for this reason that aged spores will germinate with a lower 
concentration of L-alanine than fresh spores, and in some cases 
they will germinate with alanine alone and no adenosine, or in 
other cases with adenosine alone and no L-alanine. 

Various kinds of metals have a marked effect upon germina- 
tion. Some metal ions, such as cobalt and nickel, will inhibit the 
process, whereas calcium and magnesium ions are helpful^^. We 
encountered this phenomenon in a batch of spores which had 
been produced for us in a pilot plant where the fermentors were 
made from metal. The spores we obtained from this pilot plant 
failed to germinate unless we added to the suspension some 
chelating agent such as versene or heavy concentrations of 
phosphate^'. Observations made by Brown^^ are of interest in 
this connection. He found they could germinate spores of the 
putrefactive anaerobe 3679 with versene only. In this case, it 
appeared that these spores could germinate spontaneously. 



THE BACTERIAL ENDOSPORE 35 

except for the presence of some metal ions which inhibited the 
process. Upon the addition of versene, the toxic metal ions were 
removed and germination proceeded. Dr. Riemann-^ (personal 
communication) in Denmark, working in OrdaFs laboratory, 
has made an even more interesting observation. He has been 
able to germinate most spores with an equimolecular mixture of 
calcium and dipicolinic acid. He observed this while making 
some studies on the effect of chelating agents on germination. 
Knowing that dipicolinic acid is a fairly effective chelating 
agent, he tried to use this in place of versene and found it would 
bring about germination only in the presence of calcium ions. 
The most rapid change was obtained when he had an equi- 
molecular mixture of the two. 

It is apparent from the above that the discoveries made by 
Hills can be looked upon as forerunners of many important 
advances in our knowledge of the germination of spores. 

The second observation which I mentioned above, namely, 
the demonstration of active enzymes in intact spores, was m.ade 
in connection with some of our studies on germination. We 
observed, as others have, that when spores germinate in the 
presence of L-alanine and adenosine or, as a matter of fact, with 
any of the other combinations of germ.ination ingredients, not 
all of the spores in a suspension will do so. From 90 to 98 % 
germinate, but the balance remains heat-stable and unchanged. 
The question naturally arises, why do these remaining spores 
fail to behave like the rest? Is it because they are different or 
because something has happened to the menstruum? To answer 
this question we examined the solution in which the spores had 
germinated to see whether the L-alanine or the adenosine, or 
both, had been used up. We found there appeared to be no 
change in the concentration of either, during the germination 
process. If either of the chemicals had been used, the amount 
was too small to be detected by our methods of analysis. It 
appeared nothing had happened to the solution; one would be 
tempted to assume, therefore, that the spores v/hich failed to 
germinate might be different from the remaining bulk that 

References p. 59 



36 H. O. HALVORSON 

germinated. In order to throw light upon this problem, we 
centrifuged the suspension at the conclusion of the germination 
and added to the supernatant some fresh spores, but none of 
these germinated. This clearly demonstrated that something had 
happened to the menstruum. Since there was no change in the 
total amount of alanine present, we examined the L-alanine to 
see if some had been converted to D-alanine. This appeared 
reasonable since Hills^-^ had previously demonstrated a D-alanine 
interference with germination brought about by L-alanine. A 
racemic mixture of l and d was found after less than one hour's 
contact with the spores. This prompted us to look for the 
enzyme racemase which, fortunately, was very easily found'*. 
It proved to be an interesting enzyme because it was active in 
the intact spore and was heat resistant, withstanding tempera- 
tures up to 100°. Upon more careful study, we found the enzyme 
was not only heat resistant but remained so even after the spores 
germinated. Furthermore, this enzyme was attached to some 
colloidal particles. Upon separation from the carrier by means 
of a sonic oscillation, it becomes heat sensitive. 

These observations stimulated us and others to look for more 
enzymes in spores. Previous to this time, most bacteriologists 
had assumed that spores were devoid of enzymes because most 
studies made in the past had resulted in negative findings. A 
variety of enzymes similar to the racemase have now been found 
in the spores of aerobic bacilli^-^' ^^. A heat-resistant catalase is 
present in most aerobic spores and also a heat-resistant ribo- 
sidase, an enzyme which hydrolyzes adenosine into adenine and 
ribose. The latter enzyme has also been shown to be heat 
resistant and to be associated with a colloidal particle. The heat- 
resistant catalase does not appear to be particulate. There is 
strong evidence to suspect its existence in spores of other heat- 
resistant enzymes, particularly proteolytic enzymes^'^. These 
may, in fact, be the ones responsible for the changes occurring 
during storage and also during heat shock ; for if these are like 
racemase, they will not be destroyed by the temperatures used 
for heat sensitization, and the reactions they bring about 



THE BACTERIAL ENDOSPORE 37 

may be materially speeded up at these higher temperatures. 

A variety of dormant enzymes have since been found to 
exist in spores, in addition to those mentioned above. These 
become activated when the spores germinate or are ruptured 
mechanically. In the dormant state they must be resistant to 
heat, because such enzymes can be found in spores that are 
germinated after heating. In fact, it appears from the work of 
Church and Halvorson^^ that a higher activity may be obtained 
from extract of spores following heat shock than from unheated 
spores. Although these enzymes are resistant to heat in the 
dormant state, they are heat sensitive following germination 
and mechanical rupture of the spores. The mechanisms which 
are involved in conferring heat resistance on spores also appear 
to render them inactive. I shall not attempt to discuss this 
further since it may be covered in the subsequent lecture. 

The third observation which I mentioned above, namely, 
that spores contain the chemical dipicolinic acid, has also 
proven to be a very powerful stimulus to researchers of spore 
physiology. This observation, as well as the one concerning the 
heat-resistant enzymes, was a natural consequence from Hills' 
early discovery. Powell et al.^, while studying germination, 
detected a number of organic compounds which had been 
secreted during the process. They then proceeded to examine 
the supernatants from germinated spore suspensions and, thus, 
discovered that dipicolinic acid along with other materials was 
released from the spores during germination. Among the other 
materials were calcium ions and a spore peptide. The occurrence 
of dipicolinic acid was of special interest because this was the 
first time this chemical had been reported in a natural product. 
A follow up of these studies has shown that dipicolinic acid is a 
normal constituent of all spores of bacteria (both aerobes and 
anaerobes) and that it is present in considerable quantities, 
varying from 6% to 12% of the dry weight of normal spores. 
As soon as these announcements were made, everyone who was 
interested in spore research examined their spores for this 
chemical and were able to confirm the observation of Powell. 

References p. 59 



38 H. O. HALVORSON 

Further studies on this chemical have brought out a number 
of interesting points in connection with the physiology of the 
bacterial spore. Nearly all the dipicolinic acid is released into 
the outside medium when the spores germinate. The release of 
this acid correlates almost perfectly with the loss in heat resist- 
ance, the loss in refractility, and the gain in stainability; and 
this provides good circumstantial evidence that dipicolinic acid 
plays an essential role in the unique heat-resistant properties of 
spores. Studies which have been made on the activation of the 
dormant enzymes through heat shock, germination, or mechani- 
cal rupture also show a very good correlation between the 
release of the dipicolinic acid and the activation of these 
enzymes^^. This gives further circumstantial evidence for the 
importance of dipicolinic acid in the protection of these enzymes 
in the intact spore. The acid is also released from the spores 
when they are killed by heat^^. This fact has been demonstrated 
in a number of laboratories. The temperature that is required is 
dependent upon the heat tolerance of the spores themselves. 
Thus, the spores of thermophilic organisms must be heated to 
a higher temperature to release the dipicolinic acid than those 
of some less resistant aerobic organisms. In a recent announce- 
ment by Foster^, he reports that dipicolinic acid can be released 
from spores of B. megatherium at temperatures ranging from 
70° to 100°. As the temperature is increased, less time is required. 

These numerous observations have led investigators in this 
area of study to believe that spores are made heat resistant and 
their enzymes protected by a complex collodial structure in- 
volving a polymer formed from dipicolinic acid, calcium, and 
special peptides. It would indeed be interesting to know more 
about the nature of this complex. So far, it has remained 
obscure because no means has yet been found to rupture the 
spore and retain the complex. Any form of mechanical rupture 
breaks up the complex and releases the dipicolinic acid in the 
same way as germination does. The breaking of this complex 
during germination may well be an enzymatic process and the 
enzymes may be activated by mechanical rupture as well, so that 



THE BACTERIAL ENDOSPORE 39 

when spores are broken up by mechanical means, the complex 
is again decomposed through the same mechanism that occurs 
during germination. 

The ultimate objective for most of the investigators on 
bacterial spores is to explain the means by which these structures 
gain heat resistance. To us it appears we have reached an 
impasse in our attempts to unravel this mystery by a study of 
the germination process. We have, therefore, for the time being 
at least, suspended our studies on germination and focussed our 
attention on the process of sporulation, hoping this may be a 
more fruitful study for us. I will devote the rest of this dis- 
cussion, therefore, to a report on some recent observations 
we have made on sporulation. 

Although the bulk of this report is going to be based upon 
studies made on Bacillus cereus, an aerobic sporeformer, the 
investigation had its beginnings in studies made in our labora- 
tory on the anaerobe Clostridium roseum-^. When we first began 
the study on the anaerobe, we were unable to obtain a good 
yield of spores. This was due to a recycling phenomenon 
occurring in the culture. Spores that were produced early in the 
growth cycle would germinate in the same stew in which they 
were produced. This resulted in a culture having cells in all 
stages of development including freshly germinated spores, 
actively growing vegetative cells, sporulating cells, and spores 
just released from their sporangia. In such a mixture it was 
virtually impossible to isolate clean spores. To overcome this 
difficulty we developed a technique of growing the organisms 
under semi-synchronous conditions. This was done by inocu- 
lating the medium in which the spores were to be produced with 
a very heavy inoculum from an actively growing synchronous 
culture. The result was a population in which nearly all of the 
cells began to produce spores at about the same time. We were 
thus able to harvest clean spores containing a mJnimum of 
vegetative cells and freshly germinated spores. In such cultures 
we found sporulation set in very early; in fact, the process was 
complete in about seven hours. By staining an hour or so before 

References p. 59 



40 



H. O. HALVORSON 



any dipicolinic acid was synthesized, we obtained what appeared 
to be normal spores. Furthermore, the synthesis of dipicohnic 
acid was complete, or nearly so, before any of the spores had 
developed heat resistance. Heat resistance developed approxi- 
mately an hour after the synthesis of DPA. The development of 
a spore-like structure (this study would indicate) occurs in- 
dependent of the synthesis of DPA and this precedes the 
development of heat resistance. This is shown graphically in 
Fig. 1. 

100- 



c 
'o 

-t-J 

CO 

> 
n 

S50 
o 

Q. 
0) 



Ol 




3 4 5 6 
Time in hours 



10 



Fig. 1. The relationship of heat resistance-total viable count-synthesis of 
DPA in Clostridium roseiim at 37\ O = Total viable count; • = % spore- 
stain; A = DPA synthesis; ▲ = heat resistant count. 



When we attempted to repeat this with the aerobe Bacillus 
cereus, our cultures would lyse about the time they should be 
producing spores. Investigation showed this was due to a lack 
of oxygen-i. By using a very heavy inoculum, we developed a 
condition in which the demand for oxygen exceeded our ability 
to dissolve oxygen in the water. This prompted us to make a 
study of the oxygen demand of cultures during various stages of 
growth and sporulation. The results of this study are shown in 
Fig. 2. This shows that the oxygen demand curve is bimodal. 
The first peak in this curve occurs about the time the maximum 
population of vegetative cells is reached. I should mention that 



THE BACTERIAL ENDOSPORE 



41 




^^\0a^ 








4 6 8 

Time in hours 



Fig. 2. pH, oxygen demand and residual glucose vs. time. 

the oxygen demand was determined by measuring the rate of 
disappearance of dissolved oxygen from an aliquot sample of 
the culture by means of a dropping mercury electrode and a 
polarograph. By making frequent measurements of the dissolved 
oxygen content in such a sample, we could plot a curve showing 
the change in concentration of dissolved oxygen versus time; 
and the slope of this curve, we considered the measure of the 
oxygen demand. 

As is apparent from the above, we reached the first peak in 
the oxygen demand curve at the same time we obtained the 
minimum in the pH curve. Shortly thereafter, the pH begins to 
rise and with this rise a very sharp rise m the oxygen demand 
occurs. A new enzyme system has apparently developed to 
utilize the acids which are responsible for the lowering of the 
pH. As these acids are oxidized, a very high demand for dis- 
solved oxygen develops. In fact, the oxygen demand at this 
stage far exceeds the oxygen demand during vegetative growth. 
During this second rise in the oxygen demand curve and the 
corresponding rise in the pH curve, we began to observe 



References p. 59 



42 



H. O. HALVORSON 



morphological changes in the rods which were typical of 
presporulation. Spores themselves do not actually appear until 
much later. The actual time of onset of sporulation is shown in 
Fig. 3. 




8 



12 16 

Time in hours 



Fig. 3. Time of sporulation in active culture of B. cereus T. 



We also investigated the nature of the acids which are 
released during the early vegetative cell growth and found that 
only two acids formed, pyruvic and acetic-^. The pyruvic acid 
appears first and is subsequently converted to acetic acid. This 
is illustrated in Fig. 4 and 5. These acids appear quite stable 
during the period of vegetative cell growth, but begin to dis- 
appear as the pH begins to rise and, presumably it is the 
oxidation of the acetic acid that creates the high demand for 
dissolved oxygen just preceding sporulation. Our failure to 
obtain spores in our initial experiment was because the air 
supply was not sufficient to satisfy the oxygen demand in this 
stage of the development of the culture. This difficulty can be 
overcome in one of two ways; by improving the efficiency of 
aeration, and by reducing the concentration of the glucose so 
that less acid is formed and consequently less oxygen is needed. 
This latter course also reduces the final spore crop. The meta- 
bolic processes going on during vegetative cell growth must, 
therefore, be quite different from those which take place in the 



THE BACTERIAL ENDOSPORE 



43 



acetic 
acid 








4 6 8 

Time in hours 



Fig. 4. Pyruvic and acetic acid 
production vs. time. 




Sum of pyruvic 
and acetic 
cids 



400 



300 — 

3 



200 

100 




4J 



4 6 8 

Time in hours 

Fig. 5. pH and sum of pyruvic and 
acetic acid vs. time. 



culture just preceding sporulation. During the growth of the 
vegetative cells, the glucose is broken down to acids, but the 
acids are not utilized. When the glucose is completely gone, an 
adaptive enzyme apparently forms for the utilization of the 
acids. This accounts for the rise of the pH curve. One may also 
assume that the oxidation of these acids is supplying the energy 
needed for the synthesis of the spore material. 

These observations led us to investigate a variety of inhibitors 
to see if compounds which would inhibit the utilization of inter- 
mediates might also interfere with sporulation. The first one we 
studied was a-picolinic acid"^"^. It is obvious why we selected this 
one. We beHeved it might serve as an analogue for dipicolinic 
acid and might interfere with its synthesis and thus interfere 
with the production of the spores. The results obtained with 
this inhibitor were quite surprising and are shown in Fig. 6. An 
examination of this figure will show that a-picolinic acid, when 
added to the culture while the pH is still dropping, interferes 



References p. 59 



44 



H. O. HALVORSON 




2.0x10® 
'2.0x108 



oc-picolinic acid added at 3h 
(1.2x10"5m) 



«-picolinic acid added at 2h 
(1.2 x10"5M) 



— D n D 

addition of acid 



< 10- 



4 8 12 

Time in hours 

Fig. 6. Effect of dipicolinic acid on the sporulation of B. cereus T. 

with the subsequent utilization of the acids, while the pH 
remains low and no spores result. If this material is added to the 
culture after the pH begins to rise, there is no effect; the pH 
rises normally, and the result is a normal spore crop. This 
inhibitor may, therefore, interfere with the development of the 
adaptive enzyme required for the utilization of the acetic acid. 
In view of the unexpected result with a-picolinic acid, we felt 
we should try other pyridine carboxylic acids. The results are 
shown in Table I. It is seen from this that a-picolinic acid is the 
only acid able to inhibit sporulation. 

TABLE I 

EFFECT OF PYRIDINE-CARBOXYLIC ACIDS ON THE SPORULATION OF B. CCVeUS T. 



Compound added 



Sporulation 



None -f 

Nicotinic acid (Pyridine-3-carboxylic acid) + 

Isonicotinic acid + 

Quinolinic acid + 

Pyridine-2,4-dicarboxylic acid + 

Pyridine-2,5-dicarboxylic acid + 

Dipicolinic acid (Pyridine-2.6-dicarboxylic acid) + 

a-Picolinic acid (Pyridine-2-carboxylic acid) — 



THE BACTERIAL ENDOSPORE 



45 



8 

PH 



normal 

K 7 


2.0x10 8 
^^® 7^^ 1.4 X 108 

/ Na succinate 
/ added {M/1200) 


\/ 


J. . . . 

, a-picolinic 
/ acid added 


\ J 1 /(1.2 xlO-'M) 


latest 


succinate can 


be reversing agent 



8 12 

Time in hours 



Fig. 7. Reversal by succinate of the inhibition of sporulation of B. cereiis T. 

by a-picoHnic acid. 



In Fig. 7 we show the effect of succinic acid on the inhibition 
of sporulation by a-picoHnic acid. This shows that succinic 
acid reverses the inhibition, even when it is added as late as five 
hours after growth starts. In view of this, we have tested a large 
number of other acids, including some amino acids, for their 
ability to reverse this inhibition. The results are shown in 
Table II and Table III. It is to be noted that all of the inter- 

TABLE II 

EFFECT OF AMINO ACIDS ON THE INHIBITION OF SPORULATION BY 

a-PICOLINIC ACID 



Addition of a-Picolinic acid (1.2 X 10~^ M) 1 mg/ml 
to 1 mgjml of 

Aspartic acid 

Asparagine 

Alanine 

Arginine 

CitruUine 

Cysteine 

Cystine 

Diaminopimelic acid 

Glutamic acid 

Glutamine 

Glycine 

Histidine 

/S-Hydroxyglutamic 

References p. 59 



Sporulation 

+ 
+ 



46 



H. O. HALVORSON 



TABLE III 

EFFECT OF SOME ORGANIC ACIDS ON THE INHIBITION OF SPORULATION BY 
a-PICOLINIC ACID (1.2 X 10-3 ^^ 



Compound added to a-picolinic acid 



Concentration 
mgjml 



Spondation 



Formic 

Malonic 

Propionic 

Methyl malonic 

Pimelic 

a-Ketopimelic 

Oxalic 

Adipic 

Glutamic 

Mesotartaric 

Lactic 

Pipecolic 

a-Aminopimelic 

a-Hydroxyglutaric 

DL-a-Methyl glutamic 

Glycolic 

^-Hydroxybutyric 

a-Ketoglutaric 

Fumaric 

Glyoxylic 

Pyruvic 

Acetic 

Citric 

m-Aconitic 

Isocitric 

Succinic 

Malic 

Oxalacetic 



2 

2 

2 

2 

2 

2 

2 

2 

2 

4 

1 

2 

2 

4 

1 

1 

1 

1.5 

1.5 

1.5 

0.5 

1 

1 



+ 

+ 
+ 
+ 
+ 
+ 



+ 
+ 
+ 
+ 
+ 
+ 
+ 



mediates in the tricarboxylic acid cycle and the glyoxalic acid 
shunt reverse this inhibition except fumaric acid, ketoglutaric, 
and glyoxalic acid. In addition, the inhibition is reversed with 
a number of other acids. Most of these can readily enter the 
cycles mentioned. Succinic acid proved to be the best reversing 



THE BACTERIAL ENDOSPORE 47 

agent, in that it would reverse the inhibition in concentrations 
smaller than any of the other substances tested. Among the 
amino acids, aspartic acid and asparagine are the only ones that 
were effective. 

From the above results we were led to suspect that the 
glyoxalic acid shunt was the one needed for intermediates for 
the synthesis of spore protein and dipicolinic acid. By these 
findings we were encouraged to investigate other inhibitors to 
see if we could cast further light upon this problem and get 
further indications as to whether or not the tricarboxylic acid 
cycle or the glyoxalic shunt are involved. 

Before further pursuing this problem we investigated the 
effect of metals on the inhibition with a-picolinic acid, because 
this compound is a strong chelating agent and its effect may be 
due to the removal of some essential metal ion. In order to 
interpret these experiments one needs to know the composition 
of the medium in which the spores are grown and in which the 
a-picolinic acid is producing its effect. Therefore, I indicate at 
this point the composition of the medium. This is shown in 
Table IV. Table V shows the effect of added metal ions on the 

TABLE IV 

MEDIUM USED FOR GROWTH AND SPORULATION 

OF Bacillus cereus T . 



Compound 


7o 


FeS04-7H20 


0.00005 


CuS04-5H20 


0.0005 


ZnSOi • 7H2O 


0.0005 


MnS04- H2O 


0.005 


MgS04 


0.02 


(NH4)2S04 


0.2 


CaCl2 • 2H2O 


0.08 


K2HPO4 


0.5 


Yeast extract 


0.2 


Glucose 


0.1 


Final pH 7.25-7.45. 





References p. 59 



48 H. O. HALVORSON 

TABLE V 

EFFECT OF MINERALS ON THE INHIBITION OF SPORULATION OF 

B. cereits t. by a-picoLiNic acid or versene 



Addition 


of. 


'^i-Picolinic acid 


with 


Sporulation 


4 X concn. 


of minerals 






Mn2+ 












Mg2+ 












Ca-^+ 












Fe-^+ 












Cu2+ 












Zn-- 










+ 


Co2+ 










+ 


Ni2+ 










1 


Versene (1.5 


mg ml) 








Versene. 


2 ,■ 


concn. 


of minerals 


+ 



Concentration of the minerals found in the medium, see 1 able IV. 

inhibition with a-picolinic acid. The only mineral ions that 
reverse the inhibition are zinc, cobaU, and nickel. It is to be 
noted that manganese, magnesium, calcium, iron, and copper 
do not have this effect, nor can we reverse the inhibition by 
increasing the normal minerals of the medium fourfold. From 
these data one might conclude that «-picolinic acid does bring 
about its inhibition by removing some essential ion. If this is so, 
then the ion must be bound more firmly than the ions of man- 
ganese, magnesium, calcium, iron, and copper, but less firmly 
than zinc, cobalt, and nickel ions. In the light of these results, 
we also tried versene. It is to be noticed that versene, added to 
the extent of 1.5 mg/ml, also interferes with sporulation; but the 
effect of the versene can be overcome by doubhng the concen- 
trations of the minerals which are normally present in the growth 
medium. A general chelating asent such as versene must there- 
fore have a different effect than the a-picolinic acid. If a-picolinic 
acid is producing its effect through a chelating action, it must 
have a rather specific effect upon some special mineral. It 
would be interesting to pursue this further, but in view of other 
interesting problems we have not taken the time to do so. 



THE BACTERIAL ENDOSPORE 



49 



As other possible inhibitors of sporulation, we have tried the 
esters of acids in the tricarboxylic acid cycle. The results are in- 
dicated in Table VL In this table, failure to get spores shows that 

TABLE VI 

EFFECT OF SOME ESTERS ON SPORULATION OF B. CereUS T 



Compound added 


Concentration 


Sporulation 


Ethyl pyruvate 


1.5 X 10-2 M 




Ethyl acetate 


7 X 10-2 M 


1 


Triethyl citrate 


2.4 X 10-2 ^ 


+ 


Diethyl succinate 


2 X 10-2 M 




L-Glutamic acid diethyl ester 


1 X 10-2 M 


+ 


Ethyl malonate 


1.3 X 10-2 M 




Ethyl formate 


7 X 10-2 M 


+ 


Diethyl oxalacetate 


1.2 X 10-2 M 




Ethyl propionate 


3 X 10-2 M 


+ 


None (control) 




+ 



the ester is serving as an inhibitor whereas a normal spore crop 
shows that no such inhibition takes place. Ethyl pyruvate, 
diethyl succinate, ethyl malonate, and diethyl oxalacetate in- 
hibited sporulation, but ethyl acetate, triethyl citrate, ethyl 



8 

pH 



- rnnrf**', /ml 1 




3xio8 


\ X-A-. 




\ /' 


> 
\ 


\ /' 


\ 


\ 1 addition 


X 


\ J ,o1 ester 


^> 


v'x 


7 


diethyl ^- 


normal 


malonate ^-iA 


culture 


spores/nni 




3.5 X 105 

1 1 



8 12 

Time in hours 



Fig, 8. The effect of diethyl malonate (1.3 x IO-2 M) on the pH and 
sporulation of a culture of B. cereus T. 

References p. 59 



50 



H. O. HALVORSON 



succinate, diethyl L-glutamate, ethyl formate, and ethyl pro- 
pionate did not. 

Fig. 8 shows the effect produced by ethyl malonate. It is 
obvious from this that ethyl malonate behaves differently than 
a-picolinic acid. This inhibitor prevents sporulation whether it 
is added before the pH begins to rise or afterwards. This 
inhibitor, therefore, probably does not interfere with the forma- 
tion but instead interferes with the function of some essential 
enzyme. With this inhibitor the pH rises for a while as if the 
culture were normal but finally falls to the low level produced 
with a-picolinic acid. We know from other carefully controlled 
experiments that the interference with sporulation in this case 
is not due to a drop in the pH but rather to a specific effect of 
ethyl malonate. 

Fig, 9 shows the effect produced with diethyl succinate as an 



8 

pH 



addition 


O V loS 


y'of ester 


cT^ ,-A, 


%? 


A /' ^^^^^ 


\ 


' normal / -^ 


\ / 


culture ' ^"^ 


1 / 


/ 


\ J 


,A ^diethyl 


\ / 


.,' succinate 


\^. ^. 


- 


, 







8 



12 16 

Time in hours 



Fig. 9. The effect of diethyl succinate (2 : 10"- M) on the pH and sporu- 
lation of B. cereus T. 



inhibitor. Here again, the inhibition occurs whether the inhibitor 
is added before or after the pH begins to rise. Here also, as in the 
case of the ethyl malonate, the pH rises for a while and then 
drops. Fig. 10 shows the effect produced with ethyl pyruvate. 
Here also, the inhibitor functions whether it is added before or 
after the pH begins to rise, indicating that this inhibitor, as well 



THE BACTERIAL ENDOSPORE 



51 



8 

pH 



6- 



-. ., <^R 1 


^>^^ normal / ,--A' * 
>o culture / ,^ 

\ / A' \ 


- <10'^ 


\ / ,'' ethyl pyruvate 




■ Va . 

addition of ester 
1 





4 8 12 

Time in hours 

Fig. 10. The effect of ethyl pyruvate (1.5 10- M) on the pH and sporu- 

lation of a culture of B. cereus T. 



as the other two, probably interferes with the functioning of 
some enzyme systems rather than with the production of an 
adaptive enzyme. The ethyl pyruvate acts somewhat differently 
from the two inhibitors cited above, because in this case the pH 
rises and stays high. Nevertheless, no spores are formed. We 
have also investigated the effect of various organic acids upon 
the reversal of inhibition of these ethyl esters. I am not going to 
take time to discuss the details of all these experiments, but 
suffice to say, these inhibitors were reversed by all of the inter- 
mediates in the glyoxylic acid shunt, but were not reversed by 
fumarate or other intermediates in the TCA cycle not common 
to the glyoxylic acid shunt. 

We realize it is dangerous to rely upon inhibitors alone for the 
verification of a definite pathway in a fermentation, but the 
circumstantial evidence we had for the involvement of the 
glyoxylic acid shunt led us to conduct further experiments to see 
if we could get additional support for this conclusion. We there- 
fore investigated two other possible inhibitors. If either the 
TCA or the glyoxylic shunt (the cycles are shown in Fig. 11) is 
involved, fluoroacetic acid should also be an effective inhibitor, 
inasmuch as this material interferes with the enzyme that 
converts citrate to isocitrate. We found this acid to be an 
effective inhibitor of sporulation. It did not interfere with the 

References p. 59 



52 



H. O. HALVORSON 



GLUCOSE 

♦ 

CH3-CO-COOH 

PYRUVATE 



0.1 



II 

CH3-C~S-Co A 

ACETYL CoA 



NH2-CH-COOH 

CH2-COOH 

ASPARTIC ACID 



-^ CO-COOH 

I 
CH2-COOH 

OXALACETIC /iCiD 




HO-CH-COOH 
I 
CH2-COOH 

MALIC ACID 



CH-COOH 
11 
CH-COOH 

FUMARIC ACID 



a 



CHO 

I 

COOH 

GLYOXYLIC ACID 



SPORE ^ CH2-C00H 
MATERIAL "*~ ' 



CHg-COOH 
SUCCINIC ACID 



w 



CHp-C-SCoA 

I 

CHj-COOH 

SUCCINYL CoA 



// 



COOH 



CH3-CH^ 
\ll 

C-SCoA 



METHYL MALONYL CoA 



11, 



COOH 

I 

CO 

I 

CHg 

CHg 

COOH 



CH2-COOH 

HO-C-COOH 
I 
CH2-COOH 

CITRIC ACID 



CH3-CH2 -C-SCo A 
PROPIONYL Co A 



0<- KET06LUTAHIC ACID 



COOH 

1 

CH-NHj 

CH2 

I 
CHo 

I 
COOH 

GLUTAMIC ACID 



CH3-CH2-COOH 

PROPIONIC ACID 




H-C-COOH 
II 
C-COOH 

I 
CH2-COOH 

CIS-ACOKITIC ACID 



HO-CH-COOH 
I 
CH-COOH 

I 

CH2-C00H 
ISOCITRIC ACID 




CO-COOH 
I 
CHg-COOH 



I 
CHg-COOH 

OXALOSUCCINIC ACID 



Fig. 11. TCA and glyoxylic and cycle. 



THE BACTERIAL ENDOSPORE 53 

growth of the vegetative cells. In this way, it functions very much 
like the esters we reported above. The action of this inhibitor 
was reversed by citrate, isocitrate, succinate, and malonate, but 
not by fumarate, acetate, pyruvate, or a-ketoglutarate. This is 
shown in Table VII. 

TABLE VII 

EFFECT OF SOME ORGANIC ACIDS ON THE INHIBITION OF SPORULATION 
BY FLUOROACETIC ACID (FAA) * 

Addition (final concentration 10'^ M) Viable cells/ml Heat-stable cells/ml 



None (control) 


6 X 10' 


2 X 108 


FAA only 


1.5 X 10^ 


< 100 


FAA -f Acetate 


1.3 X 10' 


2.6 X 103 


FAA + Pyruvate 


6.6 y \0^ 


1.5 X 10^ 


FAA -f Citrate 


1.4 X 108 


1.3 X 108 


FAA — Isocitrate 


2.2 X 108 


1.4 X 108 


FAA + a-Ketoglutarate 


4.8 X 10' 
(Extensive lysis) 


3.8 ' lO-' 


FAA — Succinate 


7 X 10' 


1.3 X 108 


FAA -^ Malonate 


6 X 108 


1.4 X 108 


FAA — Fumarate 


4 X 10" 


6 X 10^ 



* An active culture was used and all compounds were added immediately 
after inoculation. 



We also tried sodium bisulfite as an inhibitor, reasoning that, 
if the glyoxylic acid shunt is involved, bisulfite should tie up the 
glyoxylic acid because of its aldehyde group, and thus break the 
cycle; of course, it may also tie up the ketone group of the 
oxalacetate which is common to both the glyoxylic acid shunt 
and the TCA cycle. In any event, we found that bisulfite did 
effectively interfere with sporulation but did not interfere with 
the growth of the vegetative cells, as shown in Table VIII. This 
inhibitor was reversed by citrate, c/5-aconitate, isocitrate, suc- 
cinate, methyl malonate, malonate, and glyoxylate. It was not 
reversed by pyruvate, acetate, a-ketoglutarate. aspartate or 

References p. 59 



54 H. O. HALVORSON 



TABLE VIII 



EFFECT OF SOME ACIDS ON THE INHIBITION OF SPORULATION 
BY SODIUM BISULFITE (4 lO'^M) 

Addition of sodium bisulfite with Sporulation 



Pyruvate (10-^ M) 




Acetate (10-'^ M) 




a-Ketoglutarate (IQ-^ M) 




Aspartate (IQ-^ M) 




Malate (10- M) 


— 


Propionic acid (10 - M) 




Formic acid (10 '^ M) 




Citrate (10-^) 


+ 


m-Aconitate (lO""^ M) 


+ 


Isocitrate (10-- M) 


+ 


Succinate (10^- M) 


+ 


Methylmalonic acid (10"- M) 


+ 


Malonic acid (10"- M) 


+ 


Glyoxylic acid (lO"- M) 


+ 



malate. The reversal by glyoxylic acid and ketoglutarate is to be 
expected, since the aldehyde and ketone groups would tie up the 
bisulfite and thus remove it from the sphere of action. The fact 
that malate does not reverse the inhibition of the bisulfite may 
indicate that bisulfite is also tying up the oxalacetate and thus 
breaking the cycle at that point. 

The glyoxylic acid shunt may be needed for sporulation, but 
as yet, we do not have convincing proof. At the present time we 
are pursuing this investigation further with radioactive tracers, 
and hope, by this technique, to get conclusive proof or denial. 
Regardless of the cycle involved, all of the inhibitors we have 
studied are reversed by succinate; and succinate has proved to 
be the most effective reversing agent because it will reverse 
these inhibitors in smaller concentrations than any of the 
others. This leads us to suspect that succinic acid is an inter- 
mediate in the synthesis of spore material, and also perhaps, in 
the synthesis of DPA. There is some evidence against this 



THE BACTERIAL ENDOSPORE 55 

conclusion; in that, Martin and Foster-^, when they studied the 
incorporation of various types of labeled compounds into DPA, 
obtained little evidence for the incorporation of succinate. In 
their experiments there may have been an abundant supply of 
succinate within the cell, and therefore, it did not utilize succi- 
nate added from the outside. You may recall from data on the 
anaerobic culture, that we could separate the formation of the 
heat-sensitive spore from the synthesis of DPA and from the 
development of heat resistance. We have not been able to 
obtain such a separation in the case of the aerobes. We have, 
therefore, investigated other inhibitors to see if we could find 
some substance that would permit the synthesis of the spore 
structure but not the synthesis of DPA, and thus prevent the 
production of a heat-resistant spore. We have succeeded in 
obtaining this result with two inhibitors, ethyl oxamate and 
diethyl pimelatc. 

To pursue this study one needs some mechanism to differen- 
tiate between heat sensitive spores, vegetative cells, and germi- 
nated spores. In this case, it cannot be done by heating. Octyl 
alcohol proved to be suitable for this purpose. This alcohol is 
very toxic to vegetative cells, killing them almost instantly, and 
also will destroy germinated spores almost equally fast. Spores 
are extremely resistant to this chemical, and, as will be shown 
later, the heat sensitive spores are also resistant. Table IX shows 
the effect of octyl alcohol upon germinated spores and vegetative 
cells of B. cereus. Table X shows the effect of ethyl oxamate 

TABLE IX 

EFFECT OF OCTYL ALCOHOL ON THE VIABILITY OF SPORES, GERMINATED SPORES, 

AND VEGETATIVE CELLS OF B. C ere US T. 



Without octvl alcohol With octvl alcohol 
Type of cells ^.^^j^ ' Heat stable Viable 

Spores 3 x 10^ 2.5 x lO^ 3 x 10« 

Germinated spores 1.6 x 10^ 10^ 10^ 

Vegetative cells 6 x 10' <100 <100 

References p. 59 



56 H. O. HAL\ ORSON 

TABLE X 

EFFECT OF TIME OF ADDITION ON ETHYL OXAMATE (IQ-- M) ON THE 
PRODUCTION OF HEAT-STABLE SPORES 

Octyl alcohols-Stable Heat-stable 



Type of culture used ,, , 



cells/ml 



Spore inoculum 


2 


X 108 


6 


X 10'5 


Active culture at time 


4 


X 108 


8 


X 10^ 


Active culture pH 5.2 (falling) 


7 


X 108 


1.5 


X 105 


Active culture pH 5.8 (rising) 


6 


X 108 


6 


X 10^ 


Active culture pH 7.1 (rising) 


1.5 


X 10^ 


1.4 


X 108 


Active culture pH 7.9 (rising) 


8 


■ 10« 


2 


X 108 



upon the production of heat-resistant spores of B. cereiis. It is 
to be noted from this that ethyl oxamate interferes with the 
formation of heat-resistant spores, whether it is added in the 
beginning to a spore inoculum or an active culture, or before or 
after the pH has started to rise. If one waits, however, until the 
pH has gone up to 7.1 or higher, it has no effect. Apparently, by 
this time, the synthesis of DPA has already progressed to the 
point where heat-resistant spores can be found. We have 
examined the inhibited cultures for DPA and find there are very 
small amounts present. It is to be noted that a few heat-resistant 
spores are formed, so that the ethyl oxamate does not block the 
synthesis of DPA completely; but it does interfere with the 
synthesis sufficiently, so that more than 95 % of the spores that 
are formed are heat sensitive. The amount of DPA which is 
found in such preparations is slightly more than one would 
expect if one assumes that the heat-resistant spores have their 
normal content, and the heat-sensitive spores, none. It is 
possible, therefore, that some DPA may be present also in the 
heat sensitive spores. 

Somewhat similar results are obtained with the diethyl pime- 
late. This inhibitor, however, interferes with the development of 
normal vegetative cells if it is added to the culture at time, or 
very early in the growth of the vegetative cells. The vegetative 



THE BACTERIAL ENDOSPORE 57 

cells look abnormal, and, in fact, many of them lyse before they 
can begin to produce spores. This inhibitor may very well 
interfere with the synthesis of cell walls. If the inhibitor is added 
after the pH has started to rise (at which time the production of 
vegetative cells has been completed and presumably there is no 
further synthesis of cell wall), we find that the inhibitor does not 
interfere with the production of spores; but the spores which 
are produced are heat sensitive, as shown in Table XI. In fact, 

TABLE XI 

THE EFFECT OF TIME OF ADDITION OF DIETHYL PIMELATE (0.01 M) 

ON SPORULATION 

After 24-h incubation at 30° on shaker 

pH of culture at Viable Octyl alcohol stable Heat stable 

time of addition ( cells j ml) (cellsjml) ( cells j ml) ^ 



4.9 (falling) 


<100 


<100 


<10 


4.85 


5.3 (rising) 


10« 


1.3 X 108 


5 X 10'' 


5.4 


6.3 (rising) 


1.3 < 108 


1.7 X 108 


2.5 X 10« 


5.45 


7.3 (rising) 


1.3 X 108 


1.5 X 108 


1.5 / 106 


5.4 


7.8 (rising) 


4 X 108 


2.5 X 108 


1.5 X 106 


5.45 



For purposes of counting, cells were spun down and resuspended in 0.01 M 
phosphate buffer, pH 7.2. 

Vegetative cells and germinated spores are killed immediately on exposure 
to octyl alcohol (0.06 ml 100 ml H2O). 

the results are almost identical with those obtained with ethyl 
oxamate. Here again, more than 95 % of the spores are heat 
sensitive. These heat-sensitive spores appear to be perfectly 
normal, as far as staining is concerned. They are refractile like 
normal spores; they undergo germination with ordinary germi- 
nation nutrients, as normal spores will; and they are resistant 
to octyl alcohol. They are extremely sensitive to heat, most of 
them being killed at 65' in less than 15 min. Their heat resist- 
ance is no greater than that in vegetative cells. 

References p. 59 



58 



H. O. HALVORSON 



TABLE XII 

EFFECT OF DPA ON THE PRODUCTION OF HEAT SENSITIVE SPORES BY ETHYL 

OXAMATE OR DIETHYL PIMELATE 









Octvl alcohol-stable 




Addition 




Time of 
addition 


cells/ml 


Heat-stable 




Concn. ofoctyl alcohol 


cells/ml 








0.06 ml/ 100 ml water 




None 








5 X 108 


Ethyl oxamate 







2.5 X 108 


1.5 X 10" 


Ethyl oxamate -^ 


DPA 





3.5 X 108 


4 X 108 


Ethyl oxamate -^ 


DPA 


7 


2.2 X 108 


5 X 108 


Ethyl oxamate -- 


DPA 


9 


1 X 108 


4.5 X 108 


Diethyl pimelate 




7 


2 X 10' 


1 X 10^ 


Diethyl pimelate 


- DPA 


7 


7 X 10' 


9 X 10' 



Both of these inhibitors can be reversed by dipicolinic acid 
added from the outside. The results are shown in Table XTL It 
can be observed from this that reversal can be obtained with 
dipicolinic acid when added from the outside, as much as seven 
to nine hours later. In the presence of DPA, the spores produced 
are heat resistant. A similar experiment cannot be made with 
diethyl pimelate because this substance cannot be added to the 
cultures at the beginning. But, if this is added to the culture at 
seven hours, we find that most of the spores are heat sensitive; 
whereas if we add dipicolinic acid at the same time, all of the 
spores are heat resistant. 

To summarize our data, it indicates that the glyoxylic acid 
shunt is involved in the synthesis of spore material and dipico- 
linic acid. Some of the enzymes that are needed in this shunt 
appear not to be present in vegetative cells but are produced as 
adaptive enzymes after the sugar has been used up. Succinic 
acid appears to be important as an intermediate in the synthesis 
of the spore material, and perhaps also in the synthesis of 
dipicolinic acid. The synthesis of spore material and the produc- 
tion of a spore-like structure can occur, independent of the 



THE BACTERIAL ENDOSPORE 59 

synthesis of dipicolinic acid. The only function the dipicoHnic 
acid plays in the process is to produce a structure that can 
protect the enzymes and make the spore heat resistant. Heat 
resistance cannot develop until after the DPA has been synthe- 
sized. This lends further circumstantial evidence to the theory 
that dipicolinic acid is involved in the formation of a complex 
which serves to protect the enzymes and makes them heat 
resistant. 

ACKNOWLEDGEMENTS 

The unpublished results reported herein should not be 
credited to any one individual but to a research team in which 
the following have been my coworkers: Dr. Krishnamurty 
Gollakota*, Research Associate and Assistant Professor of 
Research; Dr. Robert Collier** and Dr. Herbert Nakata***, 
former graduate students who have completed research for 
their theses on some phase of this problem; Mr. Ivan Goldberg 
and Mr. John DePinto, current graduate students; and Mr. 
Francis Engle. laboratory assistant, and Leena Narasimhan, 
research assistant. The above individuals are to be considered 
as co-authors. 

This investigation was supported by grants from the Office 
of Naval Research and the National Institute of Health. 

REFERENCES 

1 G. M. Hills, Biochem. 7., 45 (1949) 353. 

2 N. L. Lawrence and H. O. Halvorson, /. BacterioL, 68 (1954) 334. 

3 B. T. Stewart and H. O. Halvorson, /. BacterioL, 65 (1953) 160. 

^ B. T. Stewart and H. O. Halvorson, Arch. Biochem. Biophys., 49 

(1954) 168. 
5 J. F. Powell, Biochem. J., 54 (1953) 210. 



* Present address: Director, School of Basic Sciences and Humanities, 

U.P. Agriculture University, Poolbagh (Dist. Nainital), India. 

** Present address: University of Oklahoma, Norman, Oklahoma. 

*** Present address: Department of Bacteriology & Public Health, State 

College of Washington. Pullman. Washington. 



60 H. O. HALVORSON 

J. F. Powell, Spores, Proc. A Her ton Spore Conference, Publ. No. 5, 

A.I.B.S., Washington, D.C., 1956, p. 72. 
6 J. F. Powell and R. E. Strange, Biocheni. J., 58 (1954) 80. 
' Z. J. Ordal, personal communication. 

8 E. S. Wynne, Bacteriol. Rev., 21 (1957) 259. 

9 J. W. Foster, Dipicolinic Acid and Bacterial Spores, Lecture given at the 
University of Maryland. Sponsored by American Cyanamid Co., Chas. 
Pfizer and Sons, and Merck and Co., 1959. 

1" H. O. Halvorson, The Physiology of the Bacterial 5'/70/£', Technical Uni- 
versity of Norway, A.S. Reklametrykk, Trondheim, 1958. 

^^ K. G. Gollakota, Unpublished data. 

1- G. G. K. MuRTY AND H. O. Halvorson, /. Bacteriol., 73 (1957) 230. 

1^ W. L. Brown, Thesis, University of Illinois, Urbana, 111., 1956, 

1-1 G. M. Hills, J. Gen. Microbiol., 4 (1950) 38. 

1^ N. L. Lawrence, J. Bacteriol., 70 (1955) 577. 

^^ H. M. Nakata, Thesis, University of Illinois, Urbana, 111., 1956. 

^' H. S. Levinson, Spores, Proc. Allerton Spore Conference, Publ. No. 5, 
A.I.B.S., Washington, D.C., 1957, p. 120. 

1* B. D. Church and H. Halvorson, Bacteriol. Proc. (Soc. Am. Bacteriol.), 
(1955)41. 

^^ A. Lund, Personal communication. 

■^0 R. Collier, Thesis, University of Illinois, Urbana, 111., 1958. 

"'1 H. M. Nakata, Thesis, University of Illinois, Urbana, III., 1959. 

-^ K. G. Gollakota and H. O. Halvorson, J. Bacteriol., 79 (I960) 1. 

-3 H. H. Martin and J. W. Foster, /. Bacteriol.. 76 (1958) 167. 

-^ H. RiEMANN, personal communication. 



DISCUSSION 

Kindler: Foster has claimed that diaminopimelic acid is a 
precursor of dipicoHnic acid. I wonder if diaminopimehc acid 
reversed the inhibition of a-pimehc acid, and I also would like 
to ask if there is any evidence of conversion of dipicolinic acid 
into diaminopimelic acid upon germination? 

Halvorson: The answer to your first question is that there is 
quite good evidence now that diaminopimelic acid is not a 
precursor of dipicolinic acid. Furthermore, it does not reverse 
the inhibition of a-pimelic acid. Whether dipicolinic acid can be 
converted into diaminopimelic acid has not been tested. 



THE BACTERIAL ENDOSPORE 61 

Lees: I am afraid I am totally ignorant of these matters. Does 
the spore contain structural proteins or lipoproteins, and if so 
does this same mechanism confer stability on them? Secondly 
does this mechanism you postulated for controlling heat 
stability also confer resistance to lack of water in the case of the 
spore? 

Halvorson : These abnormal spores are resistant to chemicals 
like octyl alcohol. They are not heat resistant, and like normal 
spores, do not show enzymic activity. We found a virus infecting 
our spores at a very late stage of growth; it became incorporated 
in the spore particles, and these were heat resistant. We tried 
this with our particular spores, and found them to become 
infected in the same way, with partial protection against heat. 
There were few heat-resistant spores and these contained 
dipicolinic acid. It may be that dipicolinic acid is protecting the 
virus. 

Keynan: What might be the mechanism of the protection 
given by dipicolinic acid to the spore? 

Halvorson: I wish I knew the answer to that. All we have so 
far is indirect evidence that a complex is involved, but the 
pesky complex breaks up when we rupture the spore so that 
our hands are tied and we need some new ideas before we can 
ask the right question and suggest the proper experiment. 

Keynan: Will a-dipicolinic acid inhibit sporulation in aerobic 
systems? 

Halvorson: It also does the same thing with anaerobic 
spores. Ethyl oxamate, indeed, prevents the formation of heat- 
susceptible spores in anaerobes. 

Keynan: Does ethyl pyruvate interfere with the growth of 
the vegetative cell? 

Halvorson: This is the only one of the ethyl esters tested 
that had any effect on the growth of vegetative cells. However, 
it does not stop growth altogether. It also interferes with 
germination of spores. 

Keynan: Does it interfere with sporulation or just with 
growth? 



62 H. O. HALVORSON 

Halvorson: With growth. Growth is reduced. 

Grossowicz: Is the dipicoHnic acid found inside the spore or 
on its coat? 

Halvorson: I think Dr. Gerhardt of the University of 
Michigan claimed that it is inside the spore? 

Halvorson, Jr. : Robinowmade very thin sections and noticed 
that the cortex region between the outer spore wall and the 
inner membrane had about a third of the volume of the spore. 
It showed a well defined striated structure which disappeared on 
germination. It is suggested that the dipicolinic acid lies within 
this region. Gerhardt showed, however, that dipicolinic acid 
could be removed from the cell without injuring the cortex. 

Grossowicz: You mentioned a few inhibitors of some 
energy-yielding reaction inside the spore. Is there any evidence 
of necessity for protein synthesis in order that sporulation may 
take place? 

Halvorson: Joan Powell of Porton showed that there were 
special kinds of peptides when spores germinated, and I have 
assumed all along that these represent breakdown products of 
proteins, special proteins presumably. But we have not done 
any work on this, and I am not sure that the evidence is suffi- 
cient. 

Grossowicz : What is known about chemical transformations 
occurring during the ripening or the aging of spores? 

Halvorson: We have mostly speculations. The evidence 
found in our laboratory shows that L-alanine is found in aged 
spores, but not in the supernatant of fresh spores. Krask working 
at Camp Detrick found proteolytic enzymes present in the 
intact spores. These enzymes seemed to be heat resistant. This 
would be evidence that something happens during aging and 
perhaps the same during heat shock. I assume that there is a 
small breakdown of materials necessary for germination or 
alteration of the spore walls, so that things can get in more 
easily. We do know that with aging the germination require- 
ments become simplified and the need for heat shock is reduced. 



THE BACTERIAL ENDOSPORE 63 

Hestrin: Does addition of dipicolinic acid to the non-heat- 
resistant cells confer heat stability? 

Halvorson: Cultures to which ethyl oxamate has been 
added, and which normally should yield heat-sensitive spores, 
will produce heat-resistant spores on the addition of dipicolinic 
acid. 



STUDIES ON THE GERMINATION OF SPORES OF 
BACILLUS LICHENIFORMIS 

A. KEYNAN AND M. HALMANN 

Israe institute for Biological Research, Ness-Ziona (Israel) 

Many of us will agree with Orin Halvorson's statement in a 
recent review that the 'trigger mechanism for germination is one 
of the most interesting and unique mechanisms found in 
nature'^. Although much research and speculation has been 
devoted recently to this process and akhough a picture is 
beginning to emerge of the events leading to the germination of 
bacterial spores, the exact mechanism of the latter has not yet 
been elucidated. Many of the facts connected with the 'trigger 
reaction' leading to germination have been described in the 
preceding lectures. I will, therefore, repeat only briefly some of 
those on which our conception of the nature of induction of 
germination is based. 

It is well known that freshly grown bacterial spores of many 
species will not germinate readily after harvesting. In order to 
induce germination in these spores they have to be 'aged' or 
'heat activated'. There is much evidence today that during 
'aging' or 'heat treatment' some dormant enzyme system is 
activated. Adopting Harlyn Halvorson's conception, one might 
say, that the 'heat treatment' breaks down a mechanism which 
controls dormancy. 

'Aged' or 'heat-treated' spores may be triggered readily and 
converted into heat-sensitive germinated cells by the action of a 
number of substances, among which L-alanine is the most 
common. The mechanism of the action of alanine in this 
process is not known, but both substrate and catalytic activity 
have been suggested-. O'Connor and Halvorson^ have shown 
that germination is attended by deamination of both endogenous 
and exogenous alanine by an L-alanine dehydrogenase to yield 
pyruvate. Much evidence has accumulated that metabolism of 
pyruvate is involved in germination. Inhibitors of pyruvate 



SPORE GERMINATION OF B. Ucheuiformis 65 

metabolism prevent the germination — at least in most species of 
bacteria tested. These reactions have been shown to be part of the 
metabolism of the germinating spore, but it is not yet possible 
to define which of them is responsible for the beginning of 
germination and therefore constitutes the 'prime event". 

It might be helpful in the study of this problem to find a 
system in which it would be possible to separate the 'prime 
event" ('trigger reaction") from later occurring metabolic steps. 
Working with a strain of B. licheniformis we found that 
'triggering', i.e. initiation of germination, can be brought about 
under conditions which are distinct from those under which 
germination of a 'triggered' cell can occur. Spores of this strain 
could be 'triggered' by L-alanine at temperatures above 20°, and 
after exposure to such temperatures for a short time were able 
to germinate (as manifested by a drop in optical density) even 
at a temperature as low as 0°. We interpret this to mean that the 
'trigger reaction" can occur at a temperature higher than is 
required for a subsequent m.etabolic step. UtiUsing this observa- 
tion, and with the intention of investigating the initiation of 
germination separately from its subsequent steps, an experiment 
was designed in which the heat-activated spores were preincu- 
bated with L-alanine at 37' for a few minutes, and were then 
cooled to 15° or 18°. 

As previously stated, alanine failed to initiate activation in 
cells treated directly at 15°. Nevertheless, optical density of 
spore suspensions preincubated at 37° for a few minutes con- 
tinued to decrease at 15\ The final drop in optical density at 
15° and 18° depended on the duration and temperature of 
preincubation. 

Fig. 1 presents data of such an experiment. Two minutes of 
preincubation of a spore suspension at 37° are not enough for 
germination to start after transfer to 0% but after 4 min of 
preincubation, about 30% of the spores are activated. This can 
be seen when the preincubated suspension is transferred to 0°. 

The 'prime event' starting the chain of reactions apparently 
precedes in time the measurable manifestation of germination. 

References p. 70 



66 



A. KEYNAN AND M. HALMANN 





fn'S 


37°C 


o°c 




45 




, 


a\. 


• 


• * • 


i — • r— • 


40 


- 








AS. 




35 




\ 


t +^- 








30 




\ 






+~— i — , 


4- ^^-^...^^^ 


25 

on 




1 1 1 


I 1 


• 1 


• ' 


1 1 



10 20 30 60 10 20 30 40 
A minutes B 



120 300 



Fig. 1. Decrease in optical density of a spore suspension of B. licheniformis 
at 0° after preincubation with alanine at 37' for different times. 
Conditions of experiment : Spores were washed 30 times, heat shocked for 
16 h at 60° in water, and suspended in 7 x 10- M phosphate buffer, pH 8. 
Activation by L-alanine in final concentration = 1/75 M. Total volume 
1.5 ml in test tubes of 6 mm diameter. Measurement of optical density in 
Coleman Junior spectrophometer at 540 //. A, Decrease in optical density 
of spore suspension incubated at 37 \ B, Decrease in optical density of 
spore suspension incubated at ' after preincubation at 37 \ #, preincubated 
for 0-2 min; A, preincubated for 4 min; -r, preincubated for lOmin. 



This can be shown from the preceding observations. In the 
above experiment a drop in optical density started at 37° only 
after 6 min. However, when spores which did not yet show a 
decrease in optical density after preincubation for 4 min were 
transferred to 0°, they showed germination. This means that the 
'trigger reaction' occurred some time between the second and 
the fourth minute, preceding any other measurable change. The 
fact that the induction of germination precedes any measurable 
change has been established previously by Harrell and Hal- 



SPORE GERMINATION OF B. Hchefuformis 67 

vorson'^, who demonstrated the existence of a time interval 
between a very short exposure to L-alanine and the subsequent 
manifestation of germination, and also by Woese^, who showed 
the existence of a time interval between alanine activation and 
the subsequent release of dipicolinic acid. 

All the evidence that may be derived from our experiment of 
'double temperature' exposure shows that whatever occurs at 
temperatures below 20° depends entirely on preincubation at 
the higher temperature. As already stated, extent of germina- 
tion, as measured by total drop in optical density at 15° or 18°, 
depends on duration and temperature of preincubation at 37°. 
The temperature dependence is demonstrated in Table I, which 

TABLE I 

INFLUENCE OF TEMPERATURE OF PREINCUBATION ON THE EXTENT OF GERMI- 
NATION AT 18° OF SPORE SUSPENSIONS OF B. Ucheuifonms 
(Conditions as in experiment presented in Fig. 1 ) 

Temperature of Per cent of decrease in optical Final drop in optical 
preincubation density after 6 niin of density after transfer to 

for 6 min preincubation-' ^^ ** (%) 

24° 5 

29° 2 20 

36° 19 38 



* Per cent of decrease in optical density expressed as: 

initial optical density — final optical density 

■■■,.,, ^x 100. 

initial optical density 

** The drop in optical density was considered as final when no measurable 
change occurred during 1 hour. 

gives data of an experiment in which spore suspensions were 
exposed for 6 min to different temperatures above 20° and then 
transferred to 18°. 

If we suppose that this procedure separates events occurring 
during germination, we have to conclude that whatever occurs 
at the lower temperature is an outcome, but not a part of the 

References p. 70 



68 A. KEYNAN AND M. HALMANN 

^trigger reaction" itself. Therefore, it would be interesting to 
compare the action of inhibitors on the 'alanine activation' 
during preincubation at 37° with that of the reaction occurring 
at 15\ with the idea in mind that inhibitors which prevent 
germination during preincubation at 37^ but have no influence 
on events occurring at 15° are true inhibitors of the primary 
'trigger reaction', while those preventing germination at 15° are 
inhibitors of other metabolic reactions subsequent to the 
primary triggering. 

Among inhibitors known to prevent L-alanine activation, 
D-alanine is active only if added before, together or immediately 
after addition of L-alanine. In our 'double temperature" experi- 
ment it was demonstrated that D-alanine prevented germination 
when added during preincubation at 37°, in the first stage of the 
experiment, but did not influence events at 15° when added at 
the second stage. 

Various salts inhibited germination when added during pre- 
incubation. When, however, these salts were added to spore 
suspensions after preincubation at 37° during the second stage 
of the experiment (at 15°), they were without influence on the 
rate or extent of germination. ED50 of KCl, NaNOs or LiCl 
when added together with L-alanine was found to be 8 x 10"- M. 
Some other aspects of the salt inhibhion of L-alanine activation 
may also be of interest. Salts of divalent cations were more 
active than monovalent ones: ED50 values were 5 x IQ-^ M for 
MgCl2 and 6 10^4 M for CaCl-z. The inhibiting action of the 
salts was initially reversible, since it was abolished when the 
cells were washed 10-15 min after salt addition. When, however, 
spores were exposed to the salts for as long as 2 h the germina- 
tion-inhibiting action could no longer be reversed by washing in 
a centrifuge. Inhibition by NaCl decreased with increasing 
amounts of L-alanine. The salt inhibition was therefore com- 
petitive in respect to L-alanine. 

Other inhibitors, e.g. HgCl-z and octyl alcohol prevented 
germination at whatever stage they were added, whether 
at 18° or at 37°. Presumably they block some metabolic 



SPORE GERMINATION OF B. Jicheuifonnis 69 

reaction in germination other than the primary reaction. 

The following experiment was carried out in order to learn 
whether octyl alcohol, 0.01 M acts on the 'trigger reaction' 
itself in addition to its known action on subsequent metabolic 
steps. Spores exposed to L-alanine for 10 min at 37", with and 
without the inhibitor, were washed 5 times in the cold, in order 
to remove both the alanine and the inhibitor. After washing, 
the spores were resuspended, incubated and the decrease in 
optical density measured. Those exposed to alanine alone 
germinated readily after resuspension and incubation, while 
those preincubated in L-alanine and octyl alcohol did not 
germinate (Spore counts showed that the octyl alcohol did not 
kill any of the spores). This seems to indicate that octyl alcohol 
is inhibitory both at the 'trigger reaction' and at subsequent 
metaboHc steps. Octyl alcohol is known to inhibit L-amino- 
oxidases; therefore the above observation supports the idea that 
an L-amino-oxidase might be concerned in the triggering of 
germination. In a similar experiment, ethyl pyruvate has been 
shown to inhibit L-alanine activation during preincubation at 
37° for 10 min. This appears to confirm the hypothesis that 
pyruvate metabolism is an integral part of the primary event 
leading towards germination. 

It should be noted that temperature is not the only device by 
which the 'trigger reaction' can be separated from subsequent 
metabolic steps in this strain. Another means has been provided 
by our finding that whereas L-alanine fails to activate spores at 
pH below 6.5 the spores are able to germinate in the range 
pH 5.0 to 6.5 provided that they have been triggered previously 
by L-alanine at the higher pH. 

I would like finally to raise a point of nomenclature. In this 
paper several designations have been applied to the process of 
initiation of germination, namely 'trigger reaction', 'first step', 
'prime event', 'alanine activation', and others. Might it not be 
useful to have one generally agreed designation for this process? 

In summing up, we are able to say that a prime event in the 
germination reaction has been resolved by our experiment from 

References p. 70 



70 A. KEYNAN AND M. HALMANN 

some subsequent steps involved in germination. The system 
proposed might be useful in the analysis of the sequence of 
biochemical events during the germination of bacterial spores. 

REFERENCES 

1 O. Halvorson, Bacteriol. Rev., 23 (1959) 267. 

2 W. K. Harrell and H. Halvorson, /. Bacteriol., 69 (1955) 275. 

3 R. O'Connor and H. Halvorson, /. Bacteriol., 78 (1959) 844. 

4 C. WoESE AND H. MoROWiTZ, /. BcicterioL, 76 (1958) 81. 

DISCUSSION 

Grossowicz: Does the experiment with octyl alcohol mean 
that, after what you call the 'prime event' an induction of 
enzyme synthesis takes place? 

Keynan: There is no evidence of enzyme induction, there is 
of course room for speculation. 

Harpaz: Is anything known on the effect of mercury com- 
pounds in very small doses on the stimulation of germination? 

O. Halvorson : At the concentrations we have used, the mercury 
compounds inhibited germination. 

Keynan: We tested mercury compounds and they always 
stopped germination the minute they were introduced into the 
system. Whether it affects the triggering, I do not know, but it 
certainly stops the second step. 

Hestrin: I do not quite understand the implication of the 
fact that only part of the spore population germinates depending 
on the temperature of pretreatment. 

Keynan : Some spores require lower temperatures and shorter 
times, others higher temperatures for longer times. 

O. Halvorson: I agree that individual spores have varying 
requirements for germination. 



THE BIOCHEMICAL NATURE OF THE 
DORMANT STATE IN THE BACTERIAL ENDOSPORE* 

H. HALVORSON, R. O'CONNOR AND R. DOI 

Department of Bacteriology, University of Wisconsin, Madison, Wise. 

(U.S.A.) 

The biochemical nature of the dormant state of bacterial 
endospores might seem at first to be a contradictory statement 
since the dormant state is generally associated with the absence 
of metabolic activity. This situation is particularly true of the 
bacterial endospore which is probably the most nearly inert 
biological system known. A biochemical description of the 
dormant state must include an analysis of those chemicals whose 
inclusion is essential to the production and maintenance of 
dormancy as well as the enzymic reactions available for trigger- 
ing the breaking of dormancy. 

THE CHEMICAL NATURE OF THE DORMANT STATE 

A number of quantitative chemical differences have been 
observed between spores and vegetative cells^. The spores have 
lower levels of D-amino acids, free amino acids, lipid, poly- 
saccharide, RNA and water whereas they have higher levels of 
total nitrogen, protein-bound phosphorus and labile protein- 
bound phosphorus. Our knowledge of the water content of 
spores is unsatisfactory. Henry and Friedman- reported that 
B. megatherium spores contained 58 % water whereas the 
vegetative cells contained 80% water based on weight after 
drying. Ross and Billing^, employing refractive index measure- 
ments, observed values for spores comparable with those of 
dehydrated proteins suggesting that spores are essentially 



* This investigation was supported in part by a research grant of the 
Wisconsin Alumni Research Foundation and in part by a grant from the 
Brown-Hazen Fund. 

References p. 94 



72 



H. HALVORSON et Cll. 



dehydrated. Murrell and Scott^ arrived at similar conclusions in 
considering the heat resistance of bacterial spores at various 
water activities. These same workers, however, reported that 
99 % of the volume of the spore was exchangeable with deute- 
rium-labeled water^. 

The more interesting chemical differences are those which can 
be correlated with the degree of dormancy. The first of these was 
demonstrated by Curran et al.^ who found a high level of 
calcium (5%) in bacterial spores compared to 0.54% in vegeta- 
tive cells. Sporulation in low calcium medium resulted in heat- 
sensitive spores. These findings have been confirmed by others'"^^. 
Slightly higher levels of other bivalent cations, Fq^+, Ni2+, 
Mn-+, Zn-+ and AP+, are found in spores than in vegetative 




45 60 75 

Minutes at 80° 



Fig. 1. Kinetics of heat inactivation of B. cereiis strain T spores containing 
various levels of dipicolinic acid (DPA). The numbers refer to the per cent 

dry weight of DPA. 



DORMANCY OF BACTERIAL ENDOSPORE 



73 



cells, some of which can substitute for Ca-+ in producing heat- 
resistant forms^^. 

The second correlation followed from the discovery that 
spores contain massive quantities of a chelating agent, dipi- 
colinic acid (DPA), which is absent in vegetative cells^-. This 
unique compound, which is found just prior to or coincidental 
with heat resistance, has attracted considerable interest. The 
DPA level varies from 5-15% depending on the species under 
study. The discovery several years ago that the DPA content 
could be varied by changes in the growth supplements of the 
medium!^ provided an opportunity for relating this to heat 
resistance. The rate of heat inactivation of some of these spores 
is shown in Fig. 1 . Single heat inactivation curves were observed 
with considerable variations in heat resistance. The correlation 
of their dipicolinic acid content with viability and heat resist- 
ance is shown in Fig. 2. Above 1 % DPA, the spores are viable 
and their heat resistance is proportional to DPA content. Below 



1 











Viability 




Rate of 
inactivation 



25 50 75 

fxg DPA/mg dry spores 



100 



50 



en 
(U 

s_ 
O 

a 

JD 

D 

> 



Fig. 2. Effect of the dipicolinic acid content on the viability and heat 
resistance of B. cereus strain T spores. The rate of inactivation was measured 

at 80 \ 



References p. 94 



74 H. HALVORSON et al. 

1 % DPA, viability decreases and a higher dependence of heat 
resistance on DPA is also evident. Black, Hashimoto and 
Gerhardt^, employing the technique of endotropic sporulation 
in distilled water, produced low DPA spores which were found 
to be relatively susceptible to heat. This has recently been 
extended to show a correlation between the Ca2+ content of the 
sporulation medium and DPA biosynthesis^"*. The presence of 
these two may be manditorily coupled such that Ca2+ is required 
for DPA synthesis and the latter as a means of chelating and 
maintaining high levels of Ca-+ or other divalent cations. 

RESPIRATORY ACTIVITY OF DORMANT SPORES 

The second biochemical description of spores concerns their 
apparent physiological inertness. Consider, for example, the 
aerobic sporeformers which require oxidative reactions for the 
supply of both energy and building materials. The respiratory 
rate (Qo.) on glucose for vegetative cells ranges from 60-100. 
Reports on spores have varied considerably. Levinson and 
Hyatt^^ reported a Qo, value of 2.4 in a preparation of B. 
megatherium spores containing approximately 10% germinated 
forms. Crook^^, in examining a well washed suspension of 
B. subtilis spores, observed a Qo, value of 0.3 by means of a 
microrespirometer. Unfortunately he did not report the per- 
centage of germinated forms. In any such studies the cleanliness 
of the spores and the absence of germinated forms are essential 
to measurements of the metabolic activity of the dormant forms 
themselves. 

We have reexamined this point by extensively washing fresh 
spores of B. cereus 12-16 times until they are free of detectible 
levels of germinated forms^". When dormant spores were tested 
at very high densities (30 mg/ Warburg cup) there was no 
detectible O2 uptake after 60 min in the presence of glucose, 
whereas heat-shocked spores took up 60 //I O2 and aged heat- 
shocked spores 300 [A O2. In the latter case extensive germina- 
tion is associated with respiratory activity. Based on the limita- 
tions of the manometric technique employed, the Qo, of the 



DORMANCY OF BACTERIAL ENDOSPORE 75 

1 

dormant spores is less than 0.05 or about ;r^^ of the activity 
of corresponding vegetative cells. ' 

These findings, which have been observed by a number of 
workers, illustrate several features of dormant endospores: 

1 . Their overall respiratory activity is negligible if not com- 
pletely inactive; 

2. Respiratory activity is acquired prior to germination by 
appropriate activation either by heat or by chemicals; 

3. Germination leads to more full metabolic activity. In this 
latter case, Levinson and Hyatt^^ have shown that this can 
be largely achieved under nutritional conditions where 
protein biosynthesis is negligible. It is therefore clear that 
the metabolic systems involved are pre-existent in the 
dormant spore. 

ENZYME PATTERN OF DORMANT SPORES 

It was originally believed that dormant spores were devoid of 
enzymic activity. This is clearly not the case. Since the discovery 
of alanine racemase^^ and catalase^^' -^, an increasing number of 
enzymic activities have been recognized in dormant spores. In 
some cases, such as pyrophosphatase^^ and alanine racemase^^, 
the enzyme content is higher in spores than in vegetative cells. 
A similar situation exists regarding enzymic reactions demon- 
strable in extracts of dormant spores. During the past seven 
years there has been an exponential increase in the number of 
enzymes recognizable in spores — suggesting that the enzyme 
pattern of spores may closely resemble that of the sporulating 
cell. 

Three rather broad classes of enzymes can be recognized in 
spores: 

(a) Enzymes active in intact dormant spores; 

(b) Enzymes dormant in intact spores, but recognizable 
following activation; 

(c) Enzymes active only in extracts of dormant spores. 

References p. 94 



76 H. HALVORSON et al. 

SIGNIFICANCE OF THE ENZYMIC ACTIVITIES OF DORMANT SPORES 

It is easy to imagine that the establishing of a metaboHcally 
inert dormant state can be of selective advantage to an organ- 
ism. However, one would also expect that selective pressures 
would be operative in favor of those which can survive dormancy 
and rapidly revert to vegetative forms in which the full comple- 
ment of metabolic activity is unmasked and active. The micro- 
biological literature, especially the applied aspects, are rich in 
examples of delayed dormancy. For example, McCoy and 
Hastings^'^, employing single-cell technique, found that in 
Clostridium acetobutylicurn the germination of 5 % of the freshly 
harvested spores was delayed from 11 to 117 days and one 
spore, isolated from a year-old culture, required 222 days to 
germinate. The question is raised in these examples of the 
physiological defect in these spores displaying delayed dor- 
mancy. 

A priori one can visualize two types of metabolic activity 
which can be recognized in spores: 

(1) The maintenance of a low level of metabolism for 
maintenance of the dormant state; 

(2) The maintenance of enzymes essential for the triggering 
of germination of activated spores and unmasking of 
overall metabolism. 

The question of whether or not metabolism is required, or 
even present, in dormant spores has unfortunately not been 
sufficiently considered. This problem should be subject to test in 
either dormant or delayed dormant spores. Germinated or even 
activated spores differ sufficiently in density that it is possible to 
separate clean dormant spores by density gradient sedimenta- 
tion as material for examination. If there were any appreciable 
metabolic activity present in these spores one should be able, 
for example, to demonstrate the incorporation of ^^P phosphate 
into ATP. Employing carrier-free 32PO4, and carrier isolation, 
the sensitivity of the respiratory activity should be amplified by 



DORMANCY OF BACTERIAL ENDOSPORE 77 

over a million fold. Such studies would be very interesting with 
regard to spores of varying states of dormancy. 

The second type of metabolic activity associated with the 
triggering of germination in activated spores is more readily 
subject to experimental analysis and has been in large our own 
approach to the problem. There are essentially two stages 
involved: (a) an activation reaction which may be reversible and 
(b) triggered germination of activated spores. 

ACTIVATION 

Activation serves to start the biological clock in germination 
which under suitable conditions can lead to the loss of heat 
resistance within a 10-min period. Activation can be achieved 
in a number of ways: heat shock, chemical agents, storage or 
mechanical means. The recognizable events of activation are the 
unmasking of a number of enzyme systems, the loss of some 
spore components, including DPA. disruption of the integrity 
of the exosporium and probably an increased permeability. 

Our knowledge of the sequence of events and their relative 
importance towards poising the system for germination is in- 
complete. It is not clear whether or not the process is purely 
physical or may be enzymic. Clearly more work is required in 
this direction. The activation of the lysozyme-like lytic enzyme, 
which is active against exosporium and spore walls, might 
provide an explanation. In B. megatherium-^, Mn-+-initiated 
germination can be understood by the activation of enzyme 
activity by Mn-+ and the subsequent liberation of endogenous 
germinating agents, e.g. L-alanine. 

TRIGGERING REACTIONS IN GERMINATION 

The success of germination is undoubtedly dependent upon 
the physiological competence of the activated spore. The 
enzymic nature of germination can be inferred from a number 
of considerations: 

References p. 94 



78 



H. HALVORSON et al. 



1. Germinating agents are usually normal metabolites and 
in a number of cases disappear during germination; 

2. Stereospecific binding sites can be recognized for germi- 
nating agents which are subject to competitive inhibition; 

3. The temperature dependence of germination is that ex- 
pected of an enzymic reaction^^. 25. 

4. Germination can be blocked by a number of metabolic 
poisons. 

The primary objective is the recognition of the primary 
reaction and also the metabolic reactions essential to germina- 
tion. 

We have approached this problem by characterizing the 
germinating agents of 5. cereus^^ -^. The common feature of the 
germination stimulants appears to be their biochemical relation- 
ships rather than their structural relationships. Products of 
hexose metabolism, pyruvate and its normal degradation 
products, can act as germinating agents. These findings have led 
us to examine the metabolism of germinating agents by activated 



ADENOSINE 

i 

INOSINE 






Ribose- 




R5P 



6P3*- 



GLUCOSE 

i 

Gluconate 

I 

2 KG 

I 

-2K6PG 

/ 



Xu5R 



ALANINE "in^m^^ 



G6P 



F6P 




Triose/ 

i 

P yruvate -a Lactate 

4 

Acetate 

i 

TCA cycle 



Fig. 3. The pathway of glucose oxidation in B. cereiis strain T spores. 



DORMANCY OF BACTERIAL ENDOSPORE 79 

spores and extracts of activated spores. A summary of some of the 
individual reactions demonstrated in extracts by enzyme purifi- 
cation and end product analysis are shown in Fig. 3, ref.-^. The 
compounds in large letters are primary germinating agents and 
those underlined are less effective germinating agents. Glucose 
is initially oxidized to gluconate by a soluble DPN-linked 
glucose dehydrogenase. Spores are devoid of hexokinase, 
phosphoglucomutase, phosphohexokinase and aldolase and 
subsequently lack a functional glycolytic system. Gluconate is 
converted to 2KG by a TPN-linked system which is in turn 
phosphorylated to 2K6PG by an ATP-requiring 2KG kinase. 
2K6PG is reduced in part to 6PG by a DPNH requiring 2K6PG 
reductase and in part to pyruvate by an as yet unidentified 
pathway. Spore extracts contain a complete functional hexose 
monophosphate shunt which leads to triose formation which is 
in turn converted to pyruvate. Pyruvate is oxidatively decarbox- 
ylated to acetate which is then oxidized to CO2 by a particulate 
tricarboxylic acid cycle. 

The observation that inosine serves as a more effective 
germinating agent than adenosine led to the discovery of an 
adenosine deaminase which converts adenosine to inosine and 
a heat-stable hydrolytic nucleoside ribosidase which cleaves 
inosine to the free base and ribose-^' ^9. Krask and Fulk^o have 
demonstrated in these extracts the presence of a Mg-+ activated 
ribokinase which in the presence of ATP converts ribose to 
R5P. Alternatively they found that some of the ribose is con- 
verted to RIP from adenosine by nucleoside phosphorylase 
which is converted to R5P by an active phosphoribomutase. 

The cardinal role of L-alanine in the germination of aerobic 
spores has led to a further search for its metabolism. The 
observation that L-alanine is consumed during germination-^' 
26, 31, 32 led to the demonstration that isotopically labeled 
L-alanine is converted to pyruvate and NHs^^. The relevant 
enzyme, alanine dehydrogenase, has since been isolated and 
characterized^^. 



References p. 94 



80 H. HALVORSON et al. 

PYRUVATE HYPOTHESIS 

The hypothesis that the germination agents are metabolized 
to a common intermediate which is responsible for germination, 
is supported by the above findings that pyruvate may be derived 
from alanine, adenosine or glucose. If germination requires the 
formation of products of pyruvate oxidation, one would expect 
that precursors of pyruvate would support a germination which 
was sensitive to inhibitors of pyruvate oxidation, whereas 
products of pyruvate oxidation would permit germination which 
was insensitive to these inhibitors. An example of this was 
observed for spores of B. cereus-^. Germination which normally 
occurs in the presence of glucose, pyruvate, 6PG, R5P or 2KG 
was inhibited by hexetidine, an inhibitor of pyruvate oxidation. 
The inhibition was reversed by cocarboxylase. In the presence 
of hexetidine, pyruvate and NH3 accumulate. Germination in 
the presence of acetate, however, was insensitive to hexetidine. 
Similar results have been obtained with arsenite^^. Recently 
Church^-^ has found that intermediates of the tricarboxylic acid 
cycle, fumarate, succinate, citric, and r/^-aconitic acid will 
initiate germination. One might postulate, for example, that 
germination requires energy, the formation of a-keto acids for 
amino acid synthesis or of organic acids which act as seques- 
tering agents with the heavy metals present in spores. Although 
a further clarification of this will require further experimenta- 
tion, it is clear that germination involves the initial mediation of 
energy-yielding reactions in a system characterized by a burst of 
degradative reactions. 

THE ELECTRON TRANSPORT SYSTEM AND ITS REGULATION BY DPA 

The pyruvate hypothesis, which we have just outlined, places 
also an increasing dependence of germination on the activation 
and functioning of the electron transport system of the spores. 
Since this system is essentially absent in the dormant spore, its 
activation is essential to oxidative reactions. Although one might 
a priori invoke a number of hypotheses to explain the inactive 



DORMANCY OF BACTERIAL ENDOSPORE 



81 



State of these enzymes, the approach to the problem is largely 
guided by experimental opportunities. This was provided by 
Harrell and Mantini^^ by the finding that both the glucose 
oxidizing capacity as well as the release of DPA was propor- 
tional to the length of the heat shock period. These concurrent 
activations suggested that the two phenomena were closely 
related. It was thought that during heat activation perhaps 
an enzyme inhibitor was removed or an enzyme stimulator 
released which would affect the activity of the overall respiratory 
system. 

Together with HarrelP^' ^^ we observed in extracts of heat- 
activated spores that the oxidation of glucose or of DPNH 
could be stimulated three fold by the addition of DPA, as shown 
in Fig. 4. DPA was not metabolized nor was the activity of the 



Glucose oxidation 



DPNH oxidation 




Minutes 



Fig. 4, Stimulation of glucose and DPNH oxidation by dipicolinic acid. 
The Warburg vessels contained: glycylglycine buffer, pH 7.3, 75 //moles; 
enzyme fraction with 4 mg protein; DPA as indicated and (a) glucose, 
20 //moles ;DPN, 0.7 //moles; (b) DPNH, 10 //moles. Final volume. 1.8 ml. 
Center well contained 0.2 ml of 20 °„ KOH. Incubation temperature, 30". 



References p. 94 



82 H. HALVORSON et oL 

first enzyme active on glucose, the DPN-linked glucose dehydro- 
genase, stimulated by DPA. It was apparent from these findings 
that DPA was acting in stimulating the electron transport 
system rather than at the level of substrate oxidation. Since 
DPA is a powerful chelating agent and heavy metals have been 
implicated in controlling electron transport in other systems, it 
seemed not unreasonable that it may be acting here by virtue of 
its chelating potential. Such mechanism of action was in fact 
suggested by an analysis of the DPA stimulation of the ATPase 
of spores^^. The ATPase was slightly inhibited by Mn-~ and 
perhaps certain other divalent metals present in the spore 
extract. An experiment designed to test the chelation hypothesis 
with the soluble DPNH oxidizing system was negative^^. The 
stimulation could not be attributed to the removal of an 
inhibitory metal since two other chelating agents, 8-hydroxy- 
quinoline and versene did not stimulate the enzyme, and prior 
dialysis against DPA did not abolish the DPA eifect. 

A clearer understanding of the mechanism of DPA stimula- 
tion has been handicapped by our knowledge of the electron 
transport system operative in spores. A number of observations 
have made it clear that it differs in several respects from that of 
the vegetative cells. Keilin and Hartree-^ reported that spores 
had less than 6 % of the cytochromes present in vegetative cells. 
Hachisuka et al.^^ observed similar results and also found that 
overall germination is characterized by a development in the 
respiratory system. Spencer and Powell^^, on the other hand, 
showed that the flavin content does not vary during germination. 
Nakada et alA^ observed that spores are less sensitive to cyanide 
than are vegetative cells and that germination is accompanied 
by cytochrome synthesis. 

These observations led us to a closer examination of the 
enzymes normally associated with electron transport. A com- 
parison of some of these in extracts of vegetative cells and in 
extracts of activated spores^ is shown in Table I. In vegetative 
cells DPNH oxidation is primarily associated with a particulate 
system rich in DPNH-oxidizing enzymes and cytochromes. 



DORMANCY OF BACTERIAL ENDOSPORE 



83 



TABLE I 

A COMPARISON OF ELECTRON TRANSPORT ENZYMES IN B. CereilS CELLS AND 

SPORES 



Enzyme 



Source 



Vegetative 


cell 


Spore 


spec. act. 


* 


spec, act.* 


24.9 




0.09 


0.06 




0.30 


3.9 




0.24 


7.56 




5.88 


0.36 








Particulate DPNH oxidase 

Soluble DPNH oxidase 

DPNH cytochrome c reductase 

Diaphorase 

Succinic cytochrome c reductase** 



* //Moles of DPNH oxidized/h/mg of protein. 
** /< Moles of succinate oxidized/h/mg of protein. 



Spores on the other hand have Uttle particulate activity but 
have a higher content of soluble DPNH oxidase. These findings 
as well as the cyanide-sensitive and cytochrome assays suggest 
the following electron transport systems of the two organisms 
(Fig. 5). 



DPNH- 



Pathways of electron transport 



Flavin — ^02 

Soluble 



Flavin — •-Cyt b — ^Cyt c, — ►Cyt c — ^Cyt a — ».02 

Particulate 
Flavin 



Succinic acid 



Fig. 5. The soluble and particulate electron transport system of spores and 

vegetative cells. 

References p. 94 



84 



H. HALVORSON et al. 



We have recently purified a number of these enzymes present 
in spores^. Of particular interest was the DPNH oxidase which 
is the primary route of DPNH oxidation. In Warburg studies 
with a 20-fold purified enzyme, DPA stimulated oxygen uptake 
3 fold while FMN stimulated oxygen uptake 9.4 fold. The 
lack of inhibition of the enzyme by cytochrome inhibitors, as 
well as the spectrum of the enzyme, suggest a flavoprotein 
oxidase employing FMN as a cofactor. 




K, =5.5x10" 



1.0 







Atabrine + DPA 




Km =3.1x10"^ 



10 



20 



10 



20 



xlQ- 



4x10" 



Fig. 6. Competitive inhibition of FMN and DPA by atabrine on the soluble 
DPNH oxidase. Reaction mixtures contained 0.1 M phosphate buffer, 
pH 7.3, 5 X 10-6 M atabrine, 1 10"^ M DPNH, enzyme, plus FMN and 
DPA as indicated. Optical density changes were followed at 340 m^ at 25", 



DPA and FMN appear to compete for the same site (Fig. 6). 
Atabrine, a flavine analog, competitively inhibits the stimulation 
of DPNH oxidation by either DPA or FMN. The affinity 
constant for atabrine is essentially the same calculated from 
both systems. DPA depresses the rate of FMN stimulation, this 
inhibition being reversed by higher concentrations of FMN. 

DPA thus not only can substitute for FMN in stimulating the 
enzyme but also competes with FMN for the enzyme. This 
raises the interesting speculation that DPA, which has the 



DORMANCY OF BACTERIAL ENDOSPORE 



85 



pyridine ring structure in common with DPN, may act as a 
cofactor. If it were, one would expect that an enzyme-bound 
reduced form of DPA was formed. An intermediate of this 
type. dihydrodipicoHnic acid, has recently been postulated by 
Powell and Strange-^^ who suggested the following mechanism 
for the synthesis of DPA from diketopimehc acid (Fig. 7). 



H2C^ CH2 NH3 



c c 

HOOC^ \, o^ ^COOH 



HC XH 



.C C 

HOOC-^ ^N-^ N:OOH 

H 



Qoinone 



CH 



Bacterial enzyme 



HOOC' 



^N^' 



,C 



-COOH 



a,£-Diketopimelic acid 



Dihydrodipicolinic acid 



Dipicolinic acid 



Fig. 7. Pathway of dipicolinic acid synthesis. 

Assuming that DPA can act as an electron acceptor it 
provides an explanation for a number of phenomena associated 
with dormancy : 

(1) Burst in respiratory activity of sporulation associated 
with DPA synthesis'*^; 

(2) Anaerobic germination where it may act as an electron 
sink substituting for oxygen^-; 

(3) Rise in respiration following activation and germination^^ 
by stimulating the soluble oxidase pathway-'- ^^. 

THE MECHANISM OF L-ALANINE UTILIZATION 

In the light of the previous discussion it is evident that the 
DPA-accelerated oxidation of pyruvate or products of pyruvate 
is essential to germination. The prominent role played by 
L-alanine can be understood since it represents one of the most 
direct precursors of pyruvate among the germinating agents. 
The primary event in the interaction of L-alanine with activated 
spores has thus far remained obscure. In principle, this could be 



References p. 94 



86 H. HALVORSON et Cll. 

recognized by extrapolating the metabolism of alanine to time 
zero. The collective literature on spores suggests that a specific 
receptor site exists on the spore which, following combination 
with L-alanine, leads eventually to germination. This view led us 
over 6 years ago to start a search for the identity of the L-alanine 
binding site on spores of B. cereus. The discovery of an active 
alanine racemase^^ suggested this as the active site. 

This was ruled out, however, since not only is L-alanine 
dependency on germination of various strains independent of 
alanine racemase activity, but also spore germination proceeds 
under conditions in which the enzyme is inactive^^. 

The amount of L-alanine required to initiate germination is 
small. When spores are exposed for 45 seconds to ^^C-L-alanine 
the binding of 200 molecules of L-alanine is sufficient to enable 
40% germination^-^. Later it was observed-^' ^^ that NH3 and 
pyruvate release followed L-alanine disappearance. The rate of 
NH3 release from L-alanine is dependent on heat activation 
while the amount of NH3 and pyruvate formed, when pyruvate 
oxidation is blocked by arsenite, is greater than that expected 
from the L-alanine added^^. 

In order to estimate the endogenous contribution to the 
release of NH3, the recovery of labeled NH3 from ^-^N-L-alanine 
was foUowed^^. After 10 min incubation with spores, over 90% 
of the NH3 was derived from endogenous sources. Employing 
i^C-labeled alanine and an arsenite block for pyruvate, 89 % of 
the pyruvate recovered was derived from endogenous sources. 
The equimolar pyruvate and NH3 recoveries suggested that 
compounds identical to or closely related to alanine are released. 
This was further confirmed by reisolation of the alanine from 
the medium in the absence of arsenite and demonstrating a 50% 
decrease in the specific activity of the exogenous alanine. Such 
dilutions are not unexpected since from the work of Powell and 
Strange"^^, activation and germination are accompanied by a 
depolymerization of the outer layers of spores which are rich in 
alanine. 

From the isotope studies the conversion of L-alanine to 



DORMANCY OF BACTERIAL ENDOSPORE 87 

pyruvate and NH3 is clearly established. If the deamination of 
exogenous alanine is an initial step in germination, then the 
preferential metabolism of exogenous alanine would be expected 
in the early stages of germination. This was confirmed by 
observing the liberation of I'^CO-z from Ci labeled alanine^^. The 
highest specific activity was observed initially (after only 10 min 
incubation — the earliest point at wich sufficient CO2 is produced 
to permit isolation). The findings are at least consistent with the 
view that the deamination of exogenous L-alanine precedes that 
of endogenous alanine and may be an initial step. 

NATURE OF THE INITIAL BINDING SITE FOR L-ALANINE 

The above findings suggest that the L-alanine binding site is 
the deaminating enzyme or an earlier step. If it is the enzyme 
itself, the specificity of the alanine binding site on the spore 
should be identical to that of the deaminating enzyme. To 
provide information for this comparison we have purified the 
relevant enzyme over 60 fold from activated spores and charac- 
terized it^^. The purified enzyme, an L-alanine dehydrogenase, 
carries out a DPN-linked deamination of L-alanine to pyruvate 
and ammonia. The reaction is reversible, specific for DPN, and 
has an activation energy of 8,200 cal/mole. The affinity constants 
for substrates and products as well as the other collective data 
on its properties identify the enzyme as the same as that reported 
in Bacillus subtilis vegetative cells by Pierard and Wiame^^, in 
B. cereiis vegetative cells by ourselves^^ and in Mycobacterium 
by Goldman^^. 

The amination reaction requires a proton which accounts for 
the fact that the reverse reaction is favored by more acid 
conditions. The pH optimum for deamination is high, about 
pH 10, which is in agreement with the ability of spores to ger- 
minate at high pH. 

If the L-alanine dehydrogenase is the initial step in germina- 
tion then it should be (a) inhibited by D-alanine and (b) be active 
on L-alanine analogs which act as germinating agents. In Table II 

References p. 94 



88 H. HALVORSON et al. 



TABLE II 

RATE OF AMMONIA RELASE FROM L-AMINO ACIDS 



^ . . , Rate of ammonia release 

L- Amino acids , ,, 

jumoles/h 



Alanine 


2.46 


Valine 


1.42 


Cysteine 


1.12 


Serine 


0.82 


Threonine 


0.56 


Isoleucine 


0.46 


Leucine 


0.37 



Clean, heat-shocked spores (25 mg) were incubated at 30 in Conway 
diffusion units containing 5 //moles of the indicated amino acid in 2 ml 
of 0.087 A/ phosphate buffer, pH 7.0. Corrections were made for endogenous 
(amino acid omitted) release of ammonia. Thirteen other L-amino acids 
were not deaminated. 



some of the L-amino acids which initiate gerinination and NH3 
in these spores are shown. The deamination of L-alanine as well 
as the other L-amino acids is competitively inhibited by the 
D-isomer. A survey of some of the substrate specificities of 
analogs substituted in the /j-carbon are shown in Table III. As 
can be seen the H of the /3-carbon can be substituted by alkyl 
groups or by — OH. L-alanine is itself the most active. For 
comparison purposes the germination specificities of B. subtilis 
spores studied by Woese et al.^^ are included. Although many 
parallels exist, several important differences are observed. As 
further supporting evidence that these analogs are acting on the 
same enzyme, D-alanine was found to be a competitive inhibitor 
for all of the compounds tested, and to have identical affinity 
constants to that found in inhibiting L-alanine dehydrogenation. 
Further specificities are summarized in Table IV. As can be seen 
substitutions on the a carbon lead to loss of enzyme affinity but 
not necessarily of germination. More particularly /3-NH2 com- 



DORMANCY OF BACTERIAL ENDOSPORE 



89 



TABLE III 

COMPARISON OF L-ALANINE ANALOGS AS GERMINATING AGENTS AND SUBSTRATES 

OF L-ALANINE DEHYDROGENASE 

Specificity of alanine dehydrogenase expressed as Vm determined by in- 
cubating dehydrogenase at 25" with 2 /imoles of DPN and L-alanine 
analogs, as indicated, in 0.1 M carbonate-bicarbonate buffer, pH 9.4. 
Reaction rates were measured by spectrophotometric determination of 
DPNH formation at 340 m^<. 



Substitutions on 
the ^-carbon 



% Rate of L-alanine 
Substrate Germination* 



None 


100 


CH3 


32 


CH2CH3 


17 


CH2SCH3 





CHs 
^CH3 


2.8 


CH2CH3 

■^ r->TT 


4 



CH 



— CH< 



CH3 
CH3 



< 



CHj 



OH 

—OH 

— Phenyl 



4.4 



2.5 




100 
65 
70 
12 

60 
60 
10 












* Specificity of germination taken from studies ^^ on B. subtil is germination. 

pounds are non-substrates but germinating agents. The first of 
these, /5-alanine. also does not complex with the enzyme or 
inhibit L-alanine oxidation. This is particularly important since 
Woese et alA^ observed that germination in the presence of 
/5-alanine is inhibited by D-alanine. 

These differences may be dismissed by the fact that the 
observations on germination were based on findings with 



References p. 94 



90 H. HALVORSON et al. 

TABLE IV 

COMPARISON OF L- ALANINE ANALOGS AS GERMINATING AGENTS AND SUBSTRATE 

OF L-ALANINE DEHYDROGENASE 

(Experimental procedure was the same as that for Table III) 

Substitution on the % Rate of L-alanine 

a-carbon Substrate Germination 



None 

CHs by H 
NH2 by OH 
H by CH3 


100 





100 





65 


/5 — NH2 compounds 







H 
HOOC— C— CH2— NH2 65 

H 

H 
HOOC— C— CH— CH3 75 

H NH2 

CH3 
HOOC— C— CH2— NH2 65 

H 

H 
HOOC— C— CH2— CH2— NH2 50 

H 



DORMANCY OF BACTERIAL ENDOSPORE 91 

B. subtUis while ours came from B. cereiis. We are inclined not 
to believe this, partly from the fact that the L-alanine dehydro- 
genase from B. cereus seems identical to the one described by 
Pierard and Wiame"*^ in B. subtiUs vegetative cells and also the 
evidence available with B. cereus indicates a similarity with the 
previous work on B. subtiUs. 

In considering the specificity of the initial interaction in 
germination, one point we must carefully keep in mind — that 
there are probably several alternate ways to initiate germination. 
Thus for example, L-alanine can be replaced by glucose for the 
germination of many species of spores. Also, the initial stages in 
germination involve a degradation of the exosporium liberating 
among other components L-alanine. This can be seen by blocking 
overall germination with arsenite and showing a release of 
endogenous alanine following exposure to exogenous L-alanine. 
Alanine liberated thus could act as an endogenous germinating 
agent, this germination being inhibited by D-alanine. Possibly 
this may be the mechanism of action of /i-alanine — acting as a 
stimulant for L-alanine liberation. 

The effect of these agents can be interpreted therefore in 
terms of the model shown in Fig. 8. The arguments supporting 
the L-alanine dehydrogenase as the initial binding site can be 
summarized as follows: 

1. Alanine deamination is an essential but not sufficient step 
for germination; 

2. Activation parallels activation for germination; 

3. NH3 release parallels germination; 

4. The conditions for optimal enzyme activity (high pH) are 
consistent with those for optimum germination; 

5. Both the enzyme and germination are inhibited by d- 
amino acids, especially alanine; 

6. There is a parallel specificity between enzyme and germina- 
tion. 

References p. 94 



92 



H. HALVORSON et al. 



L-ALANINE 




GERMINATING AGENTS 

Fig. 8. Route of L-alanine for germination. 












Controls (i DPA) 



30 



60 



90 120 

Minutes 



Fig. 9. Stimulatory effect of dipicolinic acid on alanine deamination by 
crude spore extracts. Dialyzed crude extract (2.1 mg protein) of heat-shocked 
spores incubated at 30 in Conway diffusion units containing 0.5 /imole 
of DPN, and, where indicated, 50 //moles of L-alanine and/or 40 /tmoles 
of DPA in 0.067 M phosphate buffer, pH 7.5. 



DORMANCY OF BACTERIAL ENDOSPORE 93 

Although the data are as yet incomplete, it is reasonably safe 
to conclude that the initial binding site is identical to or very 
similar to the L-alanine dehydrogenase. One difficulty remains 
in invoking its operation in L-alanine deamination — the equi- 
librium constant for the reaction (Kequiv. = 1.3 X lO"^^) is in 
favor of amination^^. In vegetative cells this enzyme is probably 
the route of alanine synthesis. L-alanine utilization in spores may 
yet be possible if the end products of the reaction, pyruvate and 
DPNH are rapidly utilized, thus driving the reaction to the 
right. As we have previously mentioned, pyruvate is metabolized 
by spores but the rate is low. The oxidation of DPNH by the 
DPA-stimulated soluble DPNH oxidase seems more likely. 
This was tested by measuring the rate of NH3 release from 
L-alanine in dialyzed extracts of activated spores^^. The results 
(Fig. 9) show that the rate of deamination is dramatically 
stimulated by DPA. DPA has no effect on the purified enzyme, 
and since the DPNH oxidase is present in these extracts, its role 
is undoubtedly that of recycling DPNH to DPN and thus 
keeping a low level of DPNH present in the extract. 

The release of DPA during activation, therefore, can acceler- 
ate the production of pyruvate via its stimulation of the DPNH 
oxidase and thereby produce more rapidly the products of 
pyruvate oxidation required for overall germination. 

CONCLUSION 

Our knowledge of the biochemical nature of the dormant 
state is as yet fragmentary. Some information has been presented 
indicating the role of the enzymes present in dormant and 
activated spores in converting the dormant state to the vegeta- 
tive one. One may view with optimism the possibiHty of soon 
understanding the trigger mechanism involved in breaking the 
dormant state. 



References p. 94 



94 H. HALVORSON et al. 

REFERENCES 

1 H. Halvorson and B. D. Church, Bacteriol. Rev., 21 (1957) 112, 

2 B. S. Henry and C. A. Friedman, /. Bacteriol., 33 (1937) 323. 

3 K. F. Ross AND E. Billing, /. Gen. Microbiol., 16 (1957) 418. 

4 W. G. MuRRELL AND W. J. ScoTT, Natwe, 179 (1957) 481. 

^ W. G. MuRRELL AND W. G. ScoTT, Proc. 7th Intern. Congr. Microbiol., 
Stockholm, Almquist and Wiksells, Uppsala, 1958, p. 26. 

*5 H. R. CuRRAN, B. C. Brunstetter AND A. T. Myers, /. Bacteriol., 45 
(1943) 485. 

^ S. H. Black, T. Hashimoto and P. Gerhardt, Can. J. Microbiol., 
6(1960)213. 

^ R. Doi AND H. Halvorson, Unpublished results (1960). 

9 N. Grelet, Ann. inst. Pasteur, 83 (1952) 33. 

1" R. A. Slepecky and J. W. Foster, Bacteriol. Proc, (Soc. Am. Bacterio- 
logists), 58 (1958) 43. 

11 V. ViNTER, Ceskoslov. mikrobiol, 4 (1956) 145. 

12 J. Powell, Biochem. J., 54 (1953) 210. 

13 B. D. Church and H. Halvorson, Nature, 183 (1959) 124. 
I-' C. HowiTT and H. Halvorson, Unpublished results (1960). 

15 H. S. Levinson and M. T. Hyatt, /. Bacteriol., 70 (1955) 368. 

16 P. G. Crook, /. Bacterial., 63 (1952) 193. 

1" B. D. Church and H. Halvorson, J. Bacteriol., 73 (1957) 470. 
18 B. T. Stewart and H. O. Halvorson, /. Bacteriol., 65 (1953) 160. 
1^ N. Lawrence and H. O. Halvorson, /. Bacteriol., 68 (1954) 334. 

20 W. G. Murrell, The bacterial endospore. Monograph published by the 
University of Sidney, Sidney, Australia, 1955. 

21 H. S. Levinson, J. D. Sloan Jr. and M. T. Hyatt, /. Bacteriol., 75 
(1958) 291. 

22 E. McCoy and E. G. Hastings, Proc. Soc. Expl. Biol. Med., 25 (1928) 
753. 

23 D. Keilin and F. Hartree, /. Microbiol. SeroL, 12 (1947) 115. 

-^ A. Keynan, M. Halman and Y. Avi-Dor, Proc. 7th Intern. Congr. 
Microbiol., Stockholm, Almquist and Wiksells, Uppsala, 1958, p. 37. 
25 K. Vas and G. Proszet, Nature, 779 (1957) 1301. 
2« H. Halvorson and B. D. Church, J. Appl. Bacteriol., 20 (1957) 359. 
2^ R. Doi, H. Halvorson and B. D. church, /. Bacteriol., 77 (1959) 43. 

28 N. L. Lawrence, /. Bacteriol., 70 (1955) 577. 

29 J. F. Powell and J. R. Hunter, Biochem. J., 62 (1956) 381. 

30 B. J. Krask and G. E. Fulk, Arch. Biochem. Biophys., 79 (1959) 86. 

31 G. Falcone, Giorn. Microbiol., 1 (1955) 185. 

32 G. G. K. MuRTY AND H. O. Halvorson, /. Bacteriol, 73 (1957) 233. 

33 R. O'Connor and H. Halvorson, /. Bacteriol., 78 (1959) 844. 

3^ R. O'Connor and H. Halvorson, Arch. Biochem. Biophys., 91 (1960) 290. 



DORMANCY OF BACTERIAL ENDOSPORE 95 

35 B. D. Church. Unpublished results (1959). 

3<5 W. K. Harrell and E. Mantini, Can. J. Microbiol., 3 (1957) 735. 

^' H. Halvorson, R. Doi and B. D. Church. Proc. Natl. Acad. Sci. U.S., 

44 (195S) 1171. 
3s W. K. Harrell, Can. J. Microbiol., 4 (1958) 393. 
39 R. E. Spencer and J. F. Powell, Biochem. J., 51 (1952) 239. 
^" D. Nakada, a. Matsushiro, M. Kondo, K. Suga and K. Konoshi, 

Med. J. Osaka Univ., 7 (1957) 809. 
^1 H. O. Halvorson, /. Appl. BacterioL, 20 (1957) 305. 
^^ N. G. Roth and D. H. Lively, /. BacterioL, 71 (1956) 162. 
^3 B. D. Church. H. Halvorson and H. O. Halvorson. /. BacterioL, 68 

(1954) 393. 
^-i W. K. Harrell and H. Halvorson, /. BacterioL, 69 (1955) 275. 
^5 J. F. Powell and R. E. Strange, Biochem. J., 54 (1953) 205. 
■^^ A. PiERARD AND J. M. WiAME, Biochcm. Biophvs. Acta, 37 (1960) 490. 
4' D. S. Goldman, Biochim. Biophvs. Acta, 34 (1959) 527. 
'*® C. R. WoESE, H. J. MoRowiTZ AND C. R. Hutchinson. /. BacterioL, 

75(1958)578. 
^9 Y. Hachisuka, N. Asano, M. Kaneko and T. Kanbe, Science, 124 

(1956) 174. 
50 J. F. Powell and R. E. Strange, Nature, 184 (1959) 878. 



DISCUSSION 

Keynan: Until recently it was assumed that dipicolinic acid 
was concerned with the regulation of metabolism in the spore 
stage. But Orin Halvorson reports that dipicolinic acid is not 
essential at the spore stage, since spores without dipicolinic acid 
have been obtained by him. They are, however, susceptible to 
heat. Are we to conclude then that there are several independent 
mechanisms controlling dormancy, only one of which involves 
dipicolinic acid. 

Halvorson, Jr. : It all depends on what you call a spore. The 
structures with a low dipicolinic level, which are not heat 
resistant and have high respiratory activity are not spores in our 
view. We never did produce enough of these structures to be 
able to extract enzymes, but I think they are more activated. As 
long as no proper study of the kinetics of germination of these 
structures is possible, we cannot be sure that the results involve 
any contradiction of the earlier findings. 



96 H. HALVORSON et al. 

Halvorson: There is some evidence that the heat-sensitive 
spore does not take up oxygen in the presence of glucose unless 
it is germinated with the usual germination ingredients. While 
we have not yet studied other enzyme activities, we can still 
describe it not as a heat-resistant spore but one on the way to it. 



HYPOBIOSIS IN PARASITIC WORMS 

G. WITENBERG 

Department of Parasitology, Hebrew University, Hadassah Medical School, 

Jerusalem (Israel) 

Numerous examples of temporary cessation of vital physiolo- 
gical processes are known to occur at various stages in the life 
of parasitic worms. For the most part, empirical observations 
have been recorded without any attempt to explain their 
physiological basis. True diapause, which is characterised by a 
state of obligatory rest for a certain prolonged period, apparently 
does not occur in parasitic worms. Cases of curtailment or 
cessation of activity must be regarded as phenomena of adap- 
tation which allow the egg or the larva to perform the stage 
transformation without disturbance, or the adult to survive 
unfavourable environmental conditions. This kind of crypto- 
biosis^ has usually no predetermined limit and may end as soon 
as conditions become suitable. 

EGGS 

Eggs of helminths are laid in various stages of development, 
depending on the species. In the case of undeveloped eggs, 
development may start only after they have been evacuated 
from the host and have come under the influence of atmospheric 
oxygen, given suitable conditions of temperature and other 
climatic factors, as for instance, ova of the most common human 
parasites Ascaris and TrichocephaJus. If the eggs are introduced 
into an environment lacking the necessary climatic conditions or 
free oxygen (for instance, at the bottom of fermenting sewage 
tanks) embryonic development is retarded or stops. It will 
usually be resumed when proper conditions are restored. Not all 
developmental stages of the embryo are equally adapted to such 
interruptions; generally, more advanced embryos are better able 
to withstand them. 

Capillar ia hepatica, a nematode living in the Uver-parenchyma 

References p. 106 



98 G. WITENBERG 

of small rodents, provides an instructive example of retardation 
of embryonic development. The female lays eggs incessantly, but 
they are not eliminated from the liver and accumulate in its 
tissue in large macroscopically perceptible masses. As long as the 
eggs remain in the liver they do not develop. Their development 
starts only when they are liberated after the death of the host 
(often via the droppings of the cannibalistic fellow-rodents which 
devoured the infected animal!) and come in contact with 
atmospheric oxygen. 

So called 'winter eggs' are practically unknown in helminths; 
winter interruption of embryonic development occurs in many 
species, but is caused not by any factor inherent in the egg but 
by the low temperature only. There is only one indication of 
winter eggs occurring, namely in the monogenetic trematode, 
Dactylogyrus vastatot\ living on the gills of the carp, but even 
this has been questioned recently. Nybelin- and some other 
authors supposed that this tremiatode lays 'winter eggs' which 
remain undeveloped throughout the cold season. Groeben^ 
suggested that this species lays two kinds of eggs, those which 
develop immediately, and the so called 'Dauereier' which 
develop only after some time. Bauer and Nikolskaya"* do not 
accept either of these hypotheses and assert that the cessation of 
development of the eggs of D. vastator depends on lowered 
temperature only. 

In many species of parasitic worms, especially in cestodes and 
trematodes, the newly laid ovum contains a fully developed 
larva. The larva within the egg may be motionless or it may 
move freely or rotate. Such a larva either hatches early in 
natural surroundings entirely by its own efforts, or it remains 
within the shell in a quiescent state and hatches later in the 
intestinal tract of the appropriate host. The period of survival 
of the embryonated egg outside its host varies greatly in 
different species. Eggs of some species, for instance Heterodera 
schachtii, a parasite of sugar beet, may hatch not just during one 
season but over a number of years, even when derived from a 
single batch^. 



HYPOBIOSIS IN PARASITIC WORMS 99 

In most cases, ova predestined to be swallowed are thick- 
shelled and resistant to environmental factors. They may 
survive during long periods of quiescence. The egg of the 
common human parasite Ascaris serves as an outstanding 
example of such resistance ; it can withstand most of the usual 
antiseptics and may remain alive and infective for as long as 
eight years in 4 % formalin. 

FREE LARVAE 

The hatching of larva depends on both external factors 
(temperature, enzymes of the host, etc.) and the internal 
mechanisms of the larva (pressure, specific enzymes dissolving 
the shell from within, etc.) activated by external factors. An 
interesting phenomenon occurs in the case of eggs of a plant 
nematode of the genus Meloidogyne (= Heterodera). They may 
remain quiescent in the ground for a long period and are 
stimulated to hatch by some substances produced and excreted 
by growing plant roots^. 

In many parasitic worms the infective larvae live for a varying 
period outside their host, as in the case of the miracidium and 
cercaria of trematodes, of the coracidiiim of cestodes or several 
stages of the developing larva of nematodes. However, these 
stages, except the early nematode larvae, are not fully active 
physiologically. Miracidia, cercariae and coracidia do not feed 
or develop, they move about in order to reach a suitable host. 
This is a period during which biological activity is limited in 
function and time and it may be regarded as a kind of hypo- 
biosis. Yet some of the nematode larvae, notably of the family 
Strongylidae of horses (so-called red worms) are remarkably 
vital, being able to live on the pasture twelve months or even 
longer, without change provided climatic conditions are suitable. 

In some parasitic nematodes the infective larvae crawl onto 
grasses and remain on them until they are eventually picked up 
by grazing animals'^. In some cases these larvae may slowly dry 
up intil they become fragile, surviving for long periods in this 

References p. 106 



100 G. WITENBERG 

dehydrated state. When moistened they absorb water and 
regain their normal shape and movement {Nematodirus sp., 
etc.). 

Not all species of nematodes and also not all stages of the same 
nematode have the same capacity to withstand dehydration. 
ZavadovskyS has shown that very young Trichostrongylid larvae 
cannot withstand dehydration, while five-day old ensheathed 
infective stages remain viable when dehydrated for two months 
(at 22°). On the other hand, infective larvae of the notorious 
stomach nematode of the sheep, Haemonchus contortus, perish 
rapidly when desiccated^. 

Some larvae of nematodes parasitic in plants have remarkable 
capacity to withstand desiccation. Steiner and Albin^^ recorded 
reviviscence of desiccated specimens of Anguina tritici after 28 
years and of Tylenchus polyhypnus (m.ature young females and 
larvae) after 39 years quiescence in herbarium specimens^^. It 
should be noted that the observation of the revival of dehydrated 
Anguina tritici (formerly called Anguinulina) was made as early 
as in 1743 by Needham, and is one of the earliest records of a 
cryptobiotic phenomenon. 

Free living nematodes usually show no dormancy during their 
life cycle. There are, however, coprophagous species which 
develop a so-called stage of persisting larvae ('Dauerlarve' of 
Fuchs^'"): when the larvae have consumed all available food they 
become transformed into a persisting stage which differs slightly 
morphologically from normal larvae. They are m^ore slender and 
sluggish and withstand starvation. When food again becomes 
available they start feeding and develop along normal lines. In 
some species of the genus Diplogaster which belong to the above 
mentioned group, the persisting larvae crawl onto passing dung 
beetles, hide under their elyirae and are transported to a new 
place of abundant food. They may rest in a dormant state for 
long periods on this living transport. 

Larvae of another nematode of this group, Rhabditis coarctata, 
build special elongated capsules glued to the exoskeleton of the 
dung beetles, and are carried in them until the beetle comes to a 



HYPOBIOSIS IN PARASITIC WORMS 10 1 

suitable food source, where the larvae drop, start feeding, 
become mature and multiply normally^^. 

ENCYSTED LARVAE 

The fully developed larvae of parasitic worms either enter 
actively or are transferred passively (free or included in eggs) to 
a suitable host. In the final or definitive host they develop to the 
adult stage. In the intermediate host practically all parasitic 
worms — some nematode species are excepted — encyst and 
become transformed into a higher resting larval stage. Such 
larvae of trematodes appear as metacercaria, of cestodes as 
cysticercus or plerocercoid, of acanthocephala as acanthor, of 
nematodes as agamospirura, etc. All these stages are in a state 
of dormancy which either ends when the intermediate host is 
swallowed by the definitive host, or the larvae die if quiescence 
is prolonged. The life span of the encapsuled stages of the 
helminths varies greatly; in some instances it may last for years. 
Usually the larvae encyst in a specific organ or tissue. Sometimes, 
however, they may establish themselves anywhere in the body. 
In most instances the encapsuled larvae do not change; they 
absorb just enough food from their host's tissues to maintain 
their state. In some cases, however, the encapsuled larvae grow 
and expand. The larva of the cestode Echinococcus granulosus, 
the hydatid, may develop in various mammals and in man to the 
size of a child's head and produce serious impairment of health. 

The larvae of the nematode Rhabditis maupasi* exhibit 
quiescence in peculiar circumstances. They live in their free- 
living Stage in the soil, until they are swallowed by an earth- 
worm. In the latter they partly remain active but without 
change in the protonephridia and partly encysted in various 
tissues. The latter part of the larvae apparently play the main 
role in propagation of the species. They remain quiescent 
as long as the host lives or until it autotomizes the hind 



* Syn. Rhabditis pellio sensu Buetschli, 1873, nee Schneider, 1866 (14). 
References p. 106 



102 G. WITENBERG 

part of its body in which they are concentrated^^. When the 
earthworm dies, the larvae are liberated by disintegration of its 
body and use its decaying tissues as nutrients. They maturate 
and multiply on the spot as long as the remains of the host last 
and eventually their larvae disperse to start the new life-cycle in 
another earthworm^^. 

Some encysted larvae (or their unencysted homologues living 
free in the tissues) can re-establish themselves if they are swal- 
lowed by an animal which is unsuitable as a final host. Thus, 
many insectivorous animals harbour a great number of re- 
established larvae which were originally encysted in insects; for 
instance, larvae of the dog nematode, Spirocerca lupi, larvae of 
the cat cestodes of the genus Diplopylidimu etc. Some carnivores 
may in this way acquire a great number of tetrathyridia (= 
larvae of the cestode genus Mesocestoides) which originally 
developed in mice which served as prey. Big predator fish may 
accumulate a great number of plerocercoids (= larvae of the 
cestode genus Diphyllobothrium) which originally developed in 
small fish. All these re-established larvae remain in the new host 
in their original undeveloped resting stage. 

RESTING OF NEMATODE LARVAE IN THE UNBORN FOETUS 

An organism may, under certain circumstances, acquire its 
parasites in utero. Numerous examples of prenatal infection are 
known in various animal groups. (For example, infection of 
tick's brood with piroplasms, human prenatal infection with 
toxoplasma, etc.). Several helminths may infect their hosts in 
this way. Two species of nematodes, namely, Neoascaris vitu- 
loriim in calves and Toxocara cam's in the dog, are the best known 
examples, for they show special predilection for the intrauterine 
route of infection!^. The eggs of these parasites containing 
larvae in the infective stage must be swallowed by the host; the 
larvae migrate from the intestine to the venous system and reach 
the lungs. A few burst into the alveoli of the lung and from there, 
via bronchi and trachea, they reach the pharynx where they are 



HYPOBIOSIS IN PARASITIC WORMS 103 

swallowed and reach the stomach and intestine (for the second 
time) to settle there. Dming this migration the larva moults and 
develops. When it has returned to the intestine after migration 
it is in a more advanced developmental stage than when it 
hatched from the egg. 

The majority of the larvae, however, pass through the capillary 
mesh of the lungs and enter the aiterial system. They are then 
carried to various organs and tissues where they are encapsuled, 
and eventually die. However, if the host happens to be pregnant, 
a proportion of the larvae find their way to the placenta, traverse 
it and concentrate in the lungs of the foetus. In this case an 
interesting phenomenon occurs: contrary to what happens in the 
adult host, the larvae do not move from the lung but remain 
there until the young anim.al is born. Presumably lack of oxygen, 
the necessary stimulus for the continuation of migration, prevents 
the larvae from moving. After birth, when the young animal 
begins to breathe, the larvae leave the lung and reach the 
stomach and intestine by the normal route. This interruption of 
the normal course of migration by a more or less prolonged 
resting sojourn in the lung of the foetus may be regarded as a 
kind of cryptobiosis. 

HISTOTROPIC PHASE OF NEMATODE LARVAE 

It has been long observed that in the case of some intestinal 
nematode infestations, the larvae do not develop in the lumen of 
the intestine of the definitive host, but enter the pits of the 
gastric or intestinal glands where they are sheltered and complete 
their last moulting. They then usually return to the intestinal 
lumen and develop to adults. Kotlan^^ proposed the name 
'histotropic phase' for this phenomenon. 

Numerous nematodes pass through this developmental phase, 
e.g. species of the genera Trichocephalus, Ascaridia, Trichonema, 
Hyostrongylus, Trichostrongylus, etc. In his latest paper on this 
subject, SommervilleiQ has shown that the larvae of the sheep 
nematode Ostertagia circumcincta enter the glands in the 

References p. 106 



104 G. WITENBERG 

abomasum, where they perform the last mouh, but only a part 
of them emerge immediately afterwards into the intestinal tract. 
The majority remains in the mucosa for as long as eight weeks, 
and some of them eventually die in this site without emerging 
from the nodules which have formed around them. 

The physiological significance of this phenomenon is not 
quite clear (there are nematode larvae which develop perfectly 
without it). Among several explanations proposed, it has been 
stressed-^ that this 'temporary burrowing' may have an evo- 
lutionary significance, suggesting a step in the direction of a 
more compUcated migration via the circulary system as is 
characteristic of the genera Ascaris and AncyJostoma; alter- 
natively, it may be regarded as a relict of such a migration. 

The larvae of the horse nematode, Strongylus vulgaris are 
another example of retarded histotropic phase. During their 
development they accumulate in the root of the anterior mesen- 
teric artery where they may stay for a long period and undergo 
only slight changes. The route by which the larvae reach this site 
is a matter of controversy. Their presence inside the artery is 
connected with the formation of aneurism and thrombosis which 
may cause grave consequences to the host. 

HYPOBIOSIS OF WORMS DUE TO HYPOBIOSIS OF THE HOST 

As a rule, adult parasitic worms live in uniform and constant 
conditions inside or upon their hosts and their life goes on 
uninterruptedly. In some instances, however, these conditions 
change and the parasites mostly perish. In rare cases, the parasitic 
worms are able to adapt themselves to new conditions. The most 
striking instances of such change occur during hibernation or 
aestivation of some vertebrates. Only a few relevant observations 
have been recorded. Van Beneden-^ and Markova-- observed 
changing relationships in bats, Dubinina in frogs and land 
tortoises23. 24 j^ appears that the reaction of the parasites to the 
changing physiology of the host is diff'erent in different species. 
The trematodes become letharsic until the hosts have returned 



HYPOBIOSIS IN PARASITIC WORMS 105 

to the normal state. The frog nematodes of the genus Cosmocerca, 
as well as the land tortoise nematodes of the genus Tachygonethria 
slow down their physiological processes but remain active. 

Simitch and Petrovitch-^ observed in Yugoslavia, a reaction 
of parasitic worms in hibernating spermophils CiteUus citellus. 
These mammals burrow in ground holes in the autumn to spend 
the whole winter season in sleep; however, they often wake for 
short intervals. During hibernation, their temperature falls 
approximately to that of the surroundings, but it returns to 
normal rapidly on awakening. It appears that parasitic worms 
vary in their sensitivity to these intermittent falls in temperature 
and each species is able to tolerate it for a different period. 
Some worms always die during the hibernation of the host. The 
acanthocephalan Macracanthorhynchus hinidinaceus proved to 
be the most sensitive, dying after the shortest period of reduced 
temperature in the host. This worm is normally a parasite of the 
pig and is not well adapted to the physiological conditions of the 
spermophil. The cestode, Hymenolepis nana and the nematodes 
Streptopharagus kutassi and Trichostrongylus sp. are also not 
specific parasites of the spermophil, and remain alive for no 
more than 10 days of continuous hibernation. On the other 
hand, the cestode Hymenolepis diminiita var. citelli and the 
acanthocephalan Moniliformis moniliformis can withstand 20 
days and the nematode Gongylonema longispicula even 30 days 
of continuous hibernation of the host. Encysted, i.e. resting 
larvae of two cestode species proved to be the least sensitive as 
they survived under all conditions. 

Trematode larvae developing in snails may, under certain 
circumstances, interrupt their physiological activity while their 
hosts undergo aestivation, i.e. at a relatively high temperature. 
Barbosa and Coelho-^ demonstrated that the Brasilian water 
snail, Aiistralorbis glabratus, infected with immature sporocysts 
of the human parasite. Schistosoma mansoni, is able to survive the 
dry season in a state of inactivity on the dried bottom mud for 
prolonged periods. The development of the sporocyst is then 
interrupted, but is resumed at the start of the rainy season when 

References p. 106 



106 G. WITENBERG 

the snail again becomes immersed in water. (However, if the 
sporocyst is mature, the snail dies). Similar observations were 
made by Barlow"-^ in Egypt on the snail BuUmis truncatiis 
harbouring sporocysts of Schistosoma haematobium. 

REFERENCES 

1 D. Keilin, Proc. Roy. Soc. (London), 150 (1959) 149. 

- O. Nybelin, Skr. Soedra Sverig. Fiskerifoeren, (1925) 42. 

3 G. Groeben, Z. Parasitenk., 11 (1940) 611. 

"* O. N. Bauer and N. P. Nikolskaya, Trudy Problem, i Temat. Sovesh- 

chanii, Akad. Nauk S.S.S.R., Zool. Inst., 4 (1954) 99. 
^ M. T. Franklin, Publ. Commonwealth Agr. Bur., Farnham Roval,{\95\) 

147. 
6 D. W. Fenwick, J. HelminthoL, 24 (1950) 86. 
^ K. C. Kates, Proc. Helminthol. Soc. Wash. D.C., 77 (1950) 39. 
^ M. M. Zavadovsky, Trans. Lab. Exptl. Biol. Zoo-Park Moscow, 5 (1929) 43. 
9 D. P. Furman, Am. J. Vet. Research, 5 (1944) 79. 
1" G. Steiner and F. E. Albin, J. Wash. Acad. Sci., 36 (1946) 97. 

11 T. GooDEY, /. Helminthol., 7 (1923) 47. 

12 G. Fuchs, Zool. Jahrb. Abt. Syst., 38 (1915) 109. 

1^ K. G. Leuckart, Verhcmdl. deut. zool. Ges.,L Jahresversammi, (1891) 

54. 
1^ J. K.Christie, Life History (Zooparasitica), Parasites oj Invertebrates, 

Introduction to Nematology (Ed. J. R. Christie), Sect. 2, Part 2, 1941, 

p. 246. 

15 D. Keilin, Parasitology, 17 (1925) 170. 

16 G. N. Otter, Parasitology, 25 (1933) 296. 

1" A. A. MozGOVOY, Osnovy Nematodologii, Publ. Acad. Sci. Moscow, 2 
(1953)616. 

18 A. Kotlan, Acta Vet. Hung., 7 (1949) 1. 

19 R. I. Sommerville, Australian J. Agr. Research, 5 (1954) 130. 

-° A. Chandler, J. E. Alicata and M. B. Chitwood, Life History, Intro- 
duction to Nematology, (Ed. J. R. Christie), Sect. 2, Part 2, 1941, p. 267. 

-1 P. J. VAN Beneden, Med. acad. roy. sci. Belgique, 40 (1873) 1. 

-- L. J. Markova, Zool. Zhour., 17 (1938) 133. 

"'3 M. H. DuBiNiNA, Abstr. Papers, Dept. Biol. Sci, Acad. Sci. U.S.S.R. for 
1941, (1945) quoted by Dubinlna, 1949, p. 88. 

2^ M. H. DuBiNiNA, Paras. Sbornik Zool. Inst. Acad. Sci. U.S.S.R., 11 
(1949) 61. 

-'" T. SiMiTCH AND Z. Petrovitch, Riv. parassitoL, 15 (1954) 655. 

'^^ F. S. Barbosa and M. V. Coelho, Publ. avulsas inst. Aggeu Magalhdes 
(Recife, BrazU), 4 (1955) 51. 

" C. H. Barlow, Am. J. Hyg., 22 (1935) 376. 



HYPOBIOTIC PHENOMENA IN FUNGI AND 
THEIR SIGNIFICANCE IN PLANT PATHOLOGY 

I. WAHL, 

Department oj Plant Pathology, Faculty of Agriculture, Hebrew 'Jniversity, 

Rehovot (Israel) 

The aim of this paper is to discuss some significant aspects 
of hypobiotic phenomena in fungi mainly from the plant 
pathologist's point of view. In order to define the scope of this 
discussion it seems to be necessary to clarify some basic concepts. 
It is suggested to use the term hypobiosis for resting stages in the 
life cycle of the fungus which exhibit two different biological 
phenomena. Hypobiosis can be expressed as latent existence of 
the organism, imposed by adverse environmental factors and 
lack of proper nutrients. The organisms can be easily reactivated 
by applying favorable conditions answering the requirements of 
the fungus. In this case hypobiosis constitutes an important facet 
of the protective potential of the species. In contrast to this 
concept of latent existence, Gottlieb^ and Garrett- deal primarily 
with obligate dormancy, involving only the innate state of the 
cell. A spore is dormant when it does not germinate under the 
same nutritive and environmiental influences which later allow 
production of germ tubes^. In many instances, the present state 
of knowledge does not permit distinction between these two 
processes. For this reason it is preferred to deal with the resting 
phases of the fungus life cycle as one general problem. 

Determining and understanding the resting period of the 
fungus, its duration, dependence on genetic factors and environ- 
mental conditions are of paramount importance in the control 
of plant disease by agronomic procedures such as crop rotation, 
time of planting, plowing, etc. Resting states, such as spores, 
sclerotia, resting hyphae, and rhizomorphs are important in 
enabling the pathogen to survive from one growing season to 
another. The organisms may remain in a hypobiotic stage when 
a congenial host is absent: the ideal resting state functions as 

References p. J 16 



108 I. WAHL 

a bridge between the appearance of susceptible crops. In some 
cases activation of the organisms is timed properly and secures 
persistence of the pathogen. In others, genetic and ecological 
factors may offset this timing. 'Nature's imperfection is here 
man's opportunity' -. Such an imperfection may appear in two 
ways : obligate dormancy may operate against the ability of the 
fungus to utilize suitable media that are discontinuous in their 
sequence, or on the other hand, response to stimuli in the absence 
of congenial hosts may endanger the survival of the pathogen. In 
this respect, the so-called 'decoy plants'- which stimulate the 
activation of the resting bodies but do not support their further 
perpetuation and propagation, may greatly contribute to reducing 
the potential inoculum in the soil. Introduction of these decoy 
plants into crop rotation affords an effective means for controlling 
root rots. Macfarlane^ showed that some plants resistant to the 
clubroot disease agent Plasmodium brassicae may cause ger- 
mination of its resting spores and thereby reduce their population 
in the soil. Not less interesting is the fact that oospores of the 
parasite respond to stimuli of plants taxonomically remote, such 
as Tropaeolum majus. Reseda odorata, Papaver rhoeas, Lolium 
perenne and others. 

RESTING STATES IN FUNGI 

(a) Resting spores 

Of all resting structures produced by the fungus, resting spores 
have been most thoroughly investigated. It seems to be a matter 
of general agreement^' ^ that one of the features some spores 
which exhibit long dormancy have in common is their associa- 
tion with cytological processes, such as nuclear fusion, or 
fusion and division. Oospores of Phycomycetes, ascospores of 
various fungi, teliospores of smuts and rusts, and basidiospores 
in many Hymenomycetes serve as illustrations. 

Thick-walled resting spores of Phycomycetes formed by sexual 
reproduction account for the protracted survival of the fungi in 
soil in the absence of a susceptible host. According to Schaflfnit^ 



HYPOBIOSIS IN FUNGI 109 

potato crops were attacked by the parasite Synchytrium endo- 
bioticum on field plots that had been kept fallow and free from 
weeds for more than ten years^. Oospores of Peronospora 
schleideni germinate only after several years^. Gibbs^ reported 
the survival of resting spores of Plasmodiophora brassicae for 
five years. According to Walker^, soils are known to be infested 
for ten years and longer by this parasite in the absence of the 
host plant. For these reasons crop rotation is of limited value in 
controlling this disease. Stakman and Harrar^ report that 
teliospores of Puccinia graminis formed in the late summer do 
not germinate until spring thus securing the winter survival of 
the parasite. Murphy^^ reported that some physiologic races of 
oat crown rust dilfer markedly in their precocity of telial 
development. Telial formation is hastened by adverse growing 
conditions^i. Wahl and Tobolsky (unpublished data) proved that 
oat stem rust, race two, produces telia readily on coleoptiles of 
a number of oat varieties, while races one, six and eight are 
devoid of this ability. Hingorani^^ proved that resistance of 
teliospores of P. graminis avenae to activating stimuli varies with 
physiological races. Teliospores of some races of Ustilago 
striiformis-stripe smut of several grasses requires after-ripening 
periods varying from 110 days to as long as 265 days^. 

Complications in effective disease control caused by pro- 
longed spore dormancy can be well illustrated in the case of 
wheat dwarf bunt incited by the fungus Tilletia controversa. 
While the ordinary bunt or stinking smut may be successfully 
controlled by chemical treatment of the seed, this does not hold 
true for dwarf bunt, since the spores of this fungus can remain 
in obligate dormancy in the soil for over two years^"^. It should 
be stressed that spores produced independently from the afore- 
mentioned cytological processes may also act as resting bodies 
to assure the persistence of the pathogen in an adverse environ- 
ment. Parkin found that Botrytis, Trichoderma and Stemphylium 
are capable of forming true chlamydospores under inimical 
conditions, and he concludes that the development of chlamydo- 
spores is associated with the ability to remain viable under 

References p. 116 



110 I. WAHL 

conditions which would be lethal to fungi which lack the 
capacity to produce such forms. Venkat Ram^^ cited evidence 
that antibiotics produced by bacteria induce chlamydospore 
formation in Fusarium solani. 

Several types of treatment have been employed to break 
dormancy. One of the methods is based on simulation of 
conditions prevailing in natural habitats, and consists of altering 
temperatures and humidities, a procedure widely used for rusts 
and smutsi' ^. It is postulated that by freezing and thawing, 
drying and wetting, the permeability of the spore wall becomes 
increased. Similar changes in permeability of the cell wall have 
been reported with UstUago striiformis submerged in dung 
infusion^^. Another method involves treatment with various 
chemicals — inorganic and organic acids, chloroform, ether 
benzaldehyde, siHcylaldehyde and others^' ^. In this connection, 
Stakman and Han'ar^ pose a fundamental question and 
commented upon it as follows: 'How do the various classes of 
chemical substances produce their effect? They are an aid to 
experimentation, but how does nature substitute for them? In 
some cases at least it has been shown that the effect is not on the 
permeability of the spore wall. Many of the substances are 
known to reduce the surface tension of water, and it is possible 
that they may act on the content of the spore in such a way as to 
permit greater hydration.' The author of this paper failed so far 
to induce germination in teliospores of oat stem rust despite the 
fact that water could be introduced into the spore cells. 

High temperatures have also been successfully employed in 
breaking dormancy of resting spores or chlamydospores^' i^. 
Goddard and Smith^' attribute the effect of high temperature 
shocks to activation of carboxylase. The respiratory block is 
then the inactivity of this enzyme. 

It should be pointed out that activation achieved by high 
temperature shocks can be duplicated by furfural treatment^^' i^. 
These methods achieve cell activation by putting into operation 
different biological mechanisms, e.g., heat treated spores of 
Neurospora crassa can be deactivated by exposing them to 



HYPOBIOSIS IN FUNGI 1 1 1 

aerobic conditions, while furfural-induced activation is irre- 
versible^^. 

Various nutrients have been recognized as indispensable 
factors in conditioning cell activation. Resting spores of many 
fungi are deficient in the ability to synthesize amino acids or 
vitamins and require external supply of these materials. Ryan-o 
showed that the amino acids leucine, lysine and proline brought 
about spore germination in Neurospora mutants deficient in 
these materials. 

The conidia of Glomerella cingulata have, according to Lin-^ 
special nutritional requirements for germination. A very low 
rate, or absence of germination was observed in distilled water 
or glucose solution not containing inorganic material, such as 
magnesium and phosphorus. Additional instances attesting to 
the importance of nutrients in germination have been cited by 
various authors^' "^' --' -^. There are indications that spore 
germination may be facilitated in the case of vitamin-deficient 
fungi by supplying the vitamins in question-^. The percentage 
germination of spores of a mutant strain of Fitsariwn fructi- 
genwn is correlated to the thiamine content of the spores--. 

Germination may be greatly accelerated by the presence 
of actively metabolizing plant tissues. The actively metabo- 
lizing tissue may belong to higher plants or to fungi distinctly 
different from, or the same as the fungus forming the spores 
under consideration. According to W. Brown--^, spores of 
Botrytis cinerea. Monilia fnictigena and of several other fungi 
germinate more profusely in drops of distilled water placed on 
certain plant parts of the host and non-host, than in distilled 
water alone. Leach-^ demonstrated a similar effect with the 
spores of the bean anthracnose fungus Colletotrichum Jinde- 
muthianunu and Noble-^ obtained 85 to 98% spore germination 
of Urocystis tritici with the aid of uninjured seedlings of wheat 
or the non-host rye. The activation required presoaking of the 
spores for three or four days. Christensen's-'' experiments prove 
that spores of Hehmnthosporium sativum failed to germinate 
when kept in distilled water for more than one year, but gerrni- 

References p. 116 



112 I. WAHL 

nated readily when pieces of barley tissue or sucrose were added. 

In the case of Phycomyces blakesleeanus, Robbins et al.'^^ 
contend that certain Z factors are essential for spore germina- 
tion: one of these factors has been identified as hypoxanthine. 

Fries29. 3o obtained spectacular spore germination of some 
species of Boletus and other Hymenomycetes by sowing the 
spores on malt agar with cultures of Torulopsis sanguinea. None 
of the seven species of Boletus investigated germinated on malt 
agar in the absence of the yeast organism. Fries also found 
evidence that extracts of Boletus hastened spore germination of 
the same species. 

Great difficulties have been encountered in germination of 
basidiospores of the common mushroom Psalliota bispora. 
Many attempts to induce germination in order to improve 
commercial strains by selection of monospore cultures resulted 
in failure until Ferguson^i succeeded in obtaining a high rate of 
germination by placing spores in the proximity of the growing 
mycelium. De Zeeuw^^ demonstrated later that a similar stimu- 
latory effect can be achieved if spores are transferred to agar 
media on which mycelium of the common mushroom fungus 
has been cultivated. 

To sum up the discussion on the role of germination activa- 
tors, R. Brown's^^ opinions may be of special interest. He 
advances the hypothesis that dilTerent activators operate in the 
stimulation of various dormant tissues, and that each activator 
may originate from a large number of species. At least in some 
cases the activators play an important part in the metabolism 
of the stimulated and stimulating tissue. 

Attention has been called to differences between dormancy and 
maturation-^. Stakman and Harrar^ refer to the latter phenome- 
non as apparent dormancy as contrasted with real dormancy. 
They emphasize that spores may not be ripe until they are 
liberated naturally. This was demonstrated with the basidio- 
spores o^ Pleurotus corticatus in which only naturally discharged 
spores germinated readily^. Similar observations have been 
reported with spores of Pseiidopeziza trifolii and P. medicagnis^^. 



HYPOBIOSIS IN FUNGI 1 1 3 

Cochrane-^, Garret- and Gottlieb^ do not see essential differences 
between dormancy and maturation. If the resting period is 
'relatively short', we speak of maturation; if it is, on the con- 
trary, a matter of weeks or months, the spore is said to have a 
dormant stage or resting period^. 

In some instances maturation seems to be associated with the 
completion of cytological processes, while the subsequent 
resting period involves physiological after-ripening. According 
to Blackwell-^-^, oospores of Phytophtora cactorwn are unable to 
germinate when they are first shed. Their nuclear fusion is 
delayed until three to four weeks after shedding, a further 
period of after-maturation lasting six to seven months follows 
the nuclear fusion. This period of after-ripening can be reduced 
by freezing, in contrast to the pre-fusion phase which is strongly 
fixed. 

Spores that do not need after-ripening may fail to germinate 
because of the presence of certain inhibitors liberated by the 
spore-forming fungus. Rotem (personal communication) ob- 
served that conidia of Alternaria solani do not germinate while 
attached to the conidiophores, but germinate readily even when 
artificially separated from the mycelium. Self-inhibition has 
been reported for a number of fungi, such as Uromyces phaseoli^^, 
Puccinia graminis tritici, Aspergillus niger, conidia of powdery 
mildew fungi, and other spores listed by Cochrane^. Accroding 
to Forsyth^^ uredospores of P. graminis tritici emit trimethyl- 
ethylene which inhibits spore germination. Allen^^ determined 
several properties of the substance presumably responsible for 
the inhibhion of germination in uredospores of P. graminis 
tritici. 

(b) Cryptobiosis in multicellular resting bodies 

Unlike spores consisting of one or a few cells, the resting 
bodies discussed in the following are multicellular. Their biology 
and survival ability are of paramount interest to the plant 
pathologist. Some of the pathogenic fungi capable of forming 
sclerotia, rhizomorph? or resting hyphae make disease control 

References p. 116 



114 I. WAHL 

extremely difficult^^. According to Stover^^: The majority of 
root-infecting fungi do not have an obhgate type of dormancy 
as defined by GottHeb. The 'dormancy' of most root-infecting 
fungi is enforced by adverse environmental conditions.' Table 1* 
redrawn from Stover's paper demonstrates the relationship 
between the survival ability of the reproductive resting struc- 
tures and plant rotation schedules employed for controlling 
some of the economically important diseases produced by root- 
infecting fungi. The role of microsclerotia in the persistence of 
the Verticilliiim wilt fungus was demonstrated by Wilhelm^i. He 
proved that the sclerotia of this pathogenic organism retain 
viability for 13-14 years. 

Two sclerotia-producing pathogens. Sclerotia rolfsii and 
Sclerotinia sclerotiorum cause serious losses to agricultural 
crops in Israel. 

Reduction in yields inflicted by S. rolfsii becomes very pro- 
nounced in summer. Stakman and Harrar^ stress the great 
diversity in vitamin requirements for sclerotia production 
among a relatively small number of single basidiospore isolates. 
The vitaiTiins involved are: thiamin, biotin and nicotinic acid. 

Bedi"^-' "^^ studied sclerotial production of S. sclerotiorum 
extensively. He demonstrated genetic variability among strains 
of this organism as far as sclerotial formation is concerned. 
Sclerotial formation may be stimulated by staling products 
of the same organism by low temperature and by chemicals 
such as uranium nitrate^. Sclerotial formation by a non- 
sclerotial mutant can be induced by growing it along with 
a sclerotia-producing isolate. Fully mature sclerotia floated in 
water begin to germinate after 32 days while germination com- 
mences after 16 days in sclerotia previously subjected to ultra- 
violet radiation. 



* Table I is reprinted by permission of the copyright owners, the Regents 
of the University of Wisconsin, from H. R. Stover, Plant Pathology: 
Problems and Progress, 1908-1958, the University of Wisconsin Press, 
Madison, Wise, U.S.A., 1959, p. 340. 



HYPOBIOSIS IN FUNGI 



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116 I. WAHL 

Unpublished data obtained in our laboratory show that 
production of sclerotia by laboratory cultures in Rhizoctonia' 
solani could be conspicuously increased by inoculating the 
nutrient medium with Bacillus subtilis. It was further ascertained 
by our studies that Mycosphaerella pinocles and Helmintho- 
sporium teres which incite respectively the most severe diseases of 
peas and barley in Israel, pass through the summer in this 
country as a resting mycelium. Most probably many powdery 
mildew parasites which affect various crops in Israel, as well as 
Plasmopara viticola, the cause of downy mildew on grapes and 
other important plant pathogens survive adverse conditions 
here as resting mycelia. 



REFERENCES 

1 D. Gottlieb, Botan. Rev., 16 (1950) 229. 

2 S. D. Garrett, Biology of Root-Infecting Fungi, Cambridge University 
Press, 1956. 

3 I. Macfarlane, Ann. Appl. Biol., i9 (1952) 239. 

^ V. W. Cochrane, Physiology of Fungi, John Wiley and Sons, New York, 
1958. 

5 E. ScHAFFNiT, Dent. Obstbau Ztg., 68 (1922) 212. 

6 R. McKay, Nature, 135 (1935) 306. 

' J. G. GiBBS, New Zealand J. Sci. TechnoL, A, 20 (1939) 409. 

^ J. C. Walker, Diseases of Vegetable Crops, McGraw-Hill, New York, 

1952. 
^ E. C. Stakman and J. G. Harrar, Principles of Plant Pathology, The 

Ronald Press Co., New York, 1957. 

10 H. C. Murphy, U.S. Dept. Agr. Tech. Bull., 433 (1935). 

11 M. K. Hingorani, Phytopathology, 42 (1952) 526. 

1- O. Brefeld, Mykologie, Heft 12: Hemibasidii, Muenster, 1895. 

13 D. Park, Nature, 173 (1954) 454. 

1^ C. S. Venkat Ram, Nature, 170 (1952) 889. 

15 P. C. Cheo and J. G. Leach, Phytopathology. ^0 (1950) 584. 

16 D. R. Goddard, /. Gen. Physiol., 19 (1935) 45. 

1' D. R. Goddard and P. E. Smith, Plant Physiol., 13 (1938) 241. 

18 M. A. Emerson, J. BacterioL, 55 (1948) 327. 

19 A. S. Sussman, Am. J. Botany. 40 (1953) 401. 
-0 F. J. Ryan, Am. J. Botany, 35 (1948) 497. 

-1 C. K. Lin, Am. J. Botany, 32 (1945) 296. 



HYPOBIOSIS IN FUNGI 117 

2- L. E. Hawker, Physiology of the Fungi, University of London Press, 

London, 1950. 
-3 V. G. Lilly and H. L. Burnett, Physiology of the Fungi, McGraw-Hill, 

New York. 1951. 
2-1 W. Brown, Botan. Rev., 2 (1936) 236. 

25 J. G. Leach, Minn. Univ. Agr. Expt. St a. Tech. Bull.. So. 14 (1923). 

26 R. J. Noble, J. Agr. Research, 27 (1924) 451. 

-~ J. J. Christensen, Univ. Minn. Inst. Technol. Bull., 37 (1926). 

28 W. J. RoBBiNS, V. W. Kavanagh and F. Kavanagh, Botan. Gaz., 104 
(1942) 224. 

29 N. Fries, Arch. MikrobioL, 12 (1941) 266. 

^0 N. Fries, Symbolae Botan. Upsaliensis, 6 (1943) 1. 

31 M. C. Ferguson, U.S. Dept. Agr. Bur. Plant Ind. Bull.. 16 (1902). 

32 D. J. de Zeeuw, Phytopathology, 33 (1943) 530. 

33 R. Brown, Nature, 157 (1946) 64. 

3^ F. R. Jones, U.S. Dept. Agr. Bull., 759 (1919). 

3-5 E. Blackwell, Trans. Brit. Mycol. Soc, 26 (1943) 71. 

36 C. E. Yarwood. Mycologia, 757 (1946) 64. 

3' F. R. Forsyth, Can. J. Botany, 33 (1955) 363. 

38 P. J. Allen, Phytopathology, 45 (1955) 259. 

39 L. E. Hawker. Plant Pathology. Vol. 2, Academic Press, New York, 1960. 
^" R. H. Stover. Plant Pathology: Problems and Progress, University of 

Wisconsin Press, Madison, Wise, 1959. 
-•1 S. WiLHELM. Phytopathology, 45 (1955) 180. 

42 K. S. Bedi. Indian PhytopathoL, 11 (1958) 29. 

43 K. S. Bedi. Indian PhytopathoL. 11 (1958) 110. 

DISCUSSION 

Keynan : You mentioned that spore germination occurred on 
a special variety of growing yeast : can this be replaced by yeast 
extract? 

Wahl: It may be possible, we did not try it, however, other 
yeast varieties did not give the same effect. 

Mayer: I have heard of a case of uredospores being stimulated 
by coumarin, are any other cases known? What is known of the 
properties of sclerotia, and what characterizes them as dormant 
organs? 

Wahl: Van Sumere^ and his associates ascertained that 
coumarin, o-coumaric acid, indolacetic acid and some other 
compounds cause a marked stimulation of the uredospores' 



118 I. WAHL 

germination; the mode of action of these substances is not 
clear. Your second question is rather difficult to answer, since 
different fungi are capable of producing sclerotia as a result of 
diverse stimuH. It is assumed that in many instances sclerotia 
formation is induced by inimical environmental factors. 
Sclerotia resist adverse conditions, for example Avizohar- 
Hershenzon (personal communication) demonstrated that 
sclerotia of Sclerotium rolfsii are tolerant of relatively high 
temperatures prevailing in alimentary tract of cows, but lose 
to a considerable degree their viability in compost heaps. 
Lavee'^ reported that coumarin, maleic hydrazide, and trietha- 
nolamine bring about pronounced inhibition in sclerotia 
germination of Sclerotium rolfsii and reduce the number of 
sclerotia per culture. 

Keynan: It is well known that the soil contains a large 
number of spores of fungi which will only germinate under 
favourable conditions. Inhibitors have been shown to exist in 
the soil, is anything known about these? 

Wahl: I presume that you have in mind the phenomenon 
referred to as fungistasis. Investigations in various countries 
proved that spores of many fungi rest dormant in soil but 
germinate readily when placed in distilled water. 

Dobbs and Hinson^ postulated the widespread existence of 
fungistasis in soil. The nature of the germination inhibitors is 
obscure. The inhibitory effect can be removed by specific 
organic soil amendments, by soil heating, or prolonged soil 
desiccation. Fungistatic substances can be neutralized by root 
exudates of certain plants. Buxton^' ^ found that exudates from 
the roots of pea varieties stimulate germination of spores 
belonging to fusariuni oxysporum /. pisi to which the varieties 
are vulnerable and depress that of races to which they are 
resistant. 

Chinn and Ledingham^ suggested that successful control of 
some root diseases attained by green manures application is 



HYPOBIOSIS IN FUNGI 119 

due to enhanced spore germination of the pathogen in the 
absence of the congenial host. Consequently, the produced 
hyphae perish. 



1 C. F. VAN SUMERE, C. VAN SUMERE-DE PrETER, L. C. VlNlNG AND G. A. 

Ledingham, Can. J. Microbiol., 3 (1957) 847. 

2 S. Lavee, J. Exptl. Botany, 10 (1959) 359. 

3 C. G. DoBBS AND W. H. Hinson, Nature, 172 (1953) 197. 
-i E. W. Buxton, Trans. Brit. My col. Soc, 40 (1957) 145. 

5 E. W. Buxton, Trans. Brit. Mycol. Soc, 40 (1957) 305. 

*^ S. H. F. Chinn and R. J. Ledingham, Can. J. Botany, 35 (1957) 697. 



INSECT DIAPAUSE IN RELATION TO 
THE ENVIRONMENT 

A. D. LEES 

Agricultural Research Council, Unit of Insect Physiology, Cambridge 

(Great Britain) 

The phenomenon of dormancy is a famiUar one to entomolo- 
gists. Indeed, so striking are the instances afforded by insects 
and mites that Henneguy, as long ago as 1904, appropriated the 
special term diapause ('interruption of work') to describe the 
condition. His intention was to emphasise one of the most 
noteworthy features, namely the failure of growth (embryonic, 
larval, pupal or reproductive according to species) even under 
circumstances which would be expected to favour it. Although 
the employment of the term has served to draw attention to 
ecological and physiological problems associated with dor- 
mancy, its use has not been an unmixed blessing since it has led 
some investigators to conclude that 'diapause' is a single 
phenomenon and is therefore explicable in terms of one com- 
prehensive hypothesis. Yet the examination of almost any group 
of closely related species reveals that their dormant stages occur 
at different times in the life cycle — a sure indication of their 
independent evolution. And recent studies on intraspecific 
variations have further emphasised the extraordinary plasticity 
of the diapause-controlling mechanism. With such a background 
we should be prepared for differences rather than similarities. 
'Diapause' must indeed be recognised as a portmanteau term 
covering many types of physiological arrest. 

Keihn'si classification of 'hypobiotic' forms of life is based 
on metabolic considerations. The distinction is made between 
'ametabolic' organisms which remain viable without detectable 
respiration and 'hypometabolic' organisms in which respiration 
remains appreciable, although below the level characteristic of 
active growth. In arthropods nearly all our examples of quies- 
cence and diapause fall under the latter heading. One of the rare 



INSECT DIAPAUSE AND ENVIRONMENT 121 

exceptions is provided by the larva of the Chironomid midge 
Polypedilum vanderplanki which colonizes temporary rock pools 
in northern Nigeria. In the absence of water the larvae rapidly 
become completely dehydrated. Yet, a few minutes after re- 
placing these wizened objects in water, imbibition begins, 
pharynx and heart contractions appear and the larva begins to 
swim and feed. Hinton-' ^ has shown that larvae can be desic- 
cated and rehydrated repeatedly. When thoroughly dry (con- 
taining less than 1 % water) they can be heated to 100° for short 
periods, to 65° for one day and will withstand several hours 
exposure to absolute ethanol or 77 h in liquid air. There is no 
measurable respiration. Although capable of resisting dry 
storage for over five years, they are not immortal; and after 10 
years over calcium chloride larvae often rehydrate satisfactorily 
but fail to feed and metamorphose. 

In this example dormancy is directly controlled by the 
availability of water. In other dormant or diapause states the 
relationship between the environment and the onset or termina- 
tion of the dormant state is more indirect. The relevant com- 
ponent, whether photoperiod, temperature or nutrition, acts on 
the tissues through an intermediate physiological mechanism, 
namely the endocrine system. I shall consider the question of 
receptor structures, the role of hormones in the control of 
diapause and biochemical events in the tissues in my second 
lecture. At the moment I wish to concentrate on the types of 
environmental 'cues' which are utilized by arthropods for 
controlling their dormant periods and comment briefly on the 
type of physiological information which can be secured by 
studying these external agencies. 

INDUCTION OF DIAPAUSE 

A considerable array of factors, differing according to species, 
are concerned in controlling the onset or induction of diapause. 
In temperate climates photoperiod is one of the most important. 
Many insects and mites can be said (by analogy with plants) to 

References p. 128 



122 A. D. LEES 

have a long day response, since diapause is prevented in long 
days and induced in short days. The moth Acronycta and the red 
spider mite Pcmonychus uhni belong to this group^' ^. The re- 
sponse curve shows a characteristic high rate of change at the 
critical photoperiod, which ranges from about 13 h of light 
daily in insects from southerly latitudes to 20 h in insects from 
the extreme North. In at least a few insects, however, this 
general pattern is reversed. In such 'short day' species a short 
photoperiod prevents diapause. The commercial silkworm 
Bombyx mori is the best known example. Others include the 
Desert Locust Schistocerca^ and the leafhopper Stefwcranus'^. 
It is noteworthy that the diapausing and non-diapausing forms 
of the latter species are strikingly heteromorphic. 

Since these and many other light-responsive species are 
phytophagous, the question arises as to whether the plant 
serves as the insect's photoperiodic receptor. By transferring 
them serially between different host plants, the insects or mites 
can be exposed to one type of photoperiod, their hosts to 
another. Experiments of this type have shown that in nearly all 
cases the insects respond in their own right. However, the soil- 
dwelling larvae of the cabbage root fly Erioistichia appear to be 
an exception, for although their diapause behaviour is unaffected 
by placing an opaque cover on the soil round the root of the 
plant, the light treatment of the latter is quite influential^. But 
it is not yet known whether photoperiodic or photosynthetic 
events in the plant provide the 'cue'. 

Some insight into the mode of action of the cycle of illumi- 
nation has been obtained by varying the lengths of the light and 
dark periods independently. In the mite Panonychus, for 
example, there is no doubt that both light and darkness plays 
an integral part in the photoperiodic response, the length of the 
dark period being particularly critical^. So far as the dual 
significance of the light and dark phases is concerned, other 
arthropods seem to resemble Panonychus: but the details of 
the response differ considerably. A feature which seems to be 
typical of the group (and untypical of plants) is that long 



INSECT DIAPAUSE AND ENVIRONMENT 123 

inductive dark periods are not greatly affected by short light 
breaks. The impression gained is of a photochemical reaction — 
either a synthesis or more probably a breakdown process — 
which is opposed by a dark reaction. Both the light and dark 
processes seem to develop slowly. In permanent darkness an 
intermediate response is often obtained, some individuals 
entering diapause, others developing without interruption. It 
seems possible that in these and other unnatural conditions the 
endocrine mechanism which controls development is no longer 
fully 'geared' to the photochemical receptor process and there- 
fore tends to 'drift' erratically. 

The threshold of sensitivity to light is in the region of 0.01 ft.- 
candles in many insects and is less than 0.0025 ft. -candles in the 
midge Metriocnemus investigated by Paris and Jenner^o. p^^ 
important part of the twilight in temperate latitudes therefore 
comes within the range of the photoreceptors. 

Temperature is nearly always an important factor in the control 
of diapause. As a rule high temperatures augment long photoperi- 
ods and low temperatures short ones. This means that in long day 
insects high temperatures tend to prevent diapause, while in short 
day insects, such as Bombyx, these conditions favour diapause. 

The modifying role of temperature gives some indication of 
the possible origins of obligatory and facultative diapause^^. 
Many insect varieties or sub-species differ in their diapause 
characteristics. In Bombyx, for example, there are one-genera- 
tion (univoltine) races with an obligatory diapause, 2-generation 
(bivoltine) races with a facultative diapause; and multivoltine 
races which are virtually diapause-free under any environmental 
conditions. These races may have arisen through selection of the 
temperature responses. Obviously, if the temperature at which 
the long- or short-day photoperiodic stimulus can induce a 
differential response is shifted beyond the normal developmental 
hmits, diapause would either be prevented entirely or would 
occur under all environmental conditions. A logical inference 
is that in insects with obligatory diapause the mechanism for 
perceiving these factors may still be functional. 

References p. 128 



124 A. D. LEES 

Nutrition is of occasional significance. In Panonychus for 
example, a diet of senescent leaves or foliage damaged by the 
feeding punctures of other iTiites, exerts a strong diapause- 
promoting influence and can even overcome the opposite effect 
of a long day and a high temperature. The larvae of the codling 
moth Carpocapsa ponwne/Ia are influenced photoperiodically by 
light which penetrates through the flesh of the apple; but the 
maturity of the fruit also has some effect^-^' ^^. 

Although, in general, population density has surprisingly 
little effect, crowding does sometimes induce diapause. This is 
so, for example, in the grain moth Plodia^^. 

These factors frequently act on the insect long before growth 
is actually arrested. In the case of species with a pupal diapause, 
the sensitive period may occur during larval development, the 
precise instar or instars depending on the species. Egg dor- 
mancies are controlled by the maternal physiology. However, 
the direction of the maternal response is often decided far back 
in ontogeny. In Bombyx, for example, photoperiod acts mainly 
on the late embryo, while it is still enclosed in the shell. It may 
well be that the 'directions' provided by the photoperiod are 
registered in the central nervous system and associated neuro- 
secretory centres, since these are already differentiated in the 
embryo. Perhaps even more remarkable are certain hymenop- 
teran parasites (e.g. Mormoniella) in which the diapause stage 
is the fully grown larva. The temperature conditions to which 
the mother is exposed again determine whether dormancy will 
occur. But in this instance the effect must be transmitted through 
the cytoplasm of the egg and cannot be dependent on the 
continuity of any organ system^-^. 

THE TERMINATION OF DIAPAUSE 

In many insects and mites the state of suspended animation 
is so intense that the diapausing stages finally die unless the 
appropriate releasing stimuli are forthcoming from the environ- 
ment. One gains the impression that almost any agency of 



INSECT DIAPAUSE AND ENVIRONMENT 125 

significance in the natural environment can be utilized by 
natural selection as an appropriate signal. Thus the hypopus 
of the mite Histiostoma (this is the extra non- feeding nymphal 
instar which appears when poor nutritive conditions supervene) 
can be induced to moult and transform into a feeding nymph by 
the smell of yeast^^. 

However, temperature is undoubtedly of very general signi- 
ficance. The silkworm Qgg — in this respect a classical object of 
study — has a temperature optimum for the completion of 
diapause of about 8°, which is well below the threshold for 
normal development. The temperatures required by different 
species show a general correlation with their environments, 
those from temperate or cold climates usually needing a tempera- 
ture in the range 0-10°, while 10-15' is often more suitable for 
species from warmer regions. Higher temperatures are also 
effective in insects from temperate climates which have an 
aestivating rather than a winter dormancy {e.g. the winter moth 
Operophtera^'^). 

The precise temperature requirements for terminating dia- 
pause are complex and variable. No two species are exactly alike 
in this respect. The silkworm egg needs 40 or so days of chilling. 
But longer or shorter periods may be required by other species 
with a diapause of greater or lesser intensity. It is also very 
common to find that the temperature optimum varies during the 
course of diapause completion. I shall consider some of the 
physiological implications of this phenomenon in my second 
lecture. 

DORMANCY AND THE LIFE CYCLE 

Although it is not always possible to estim.ate the precise 
significance of the m.ore complex temperature requirem.ents, it 
is safe to infer that they represent a successful adjustment to the 
average temperature conditions of the local environment. A 
dormant period of optimal length ensures that the active growth 
of the population is delayed until weather and food supplies 

References p. 128 



126 A. D. LEES 

are most favourable. Nevertheless, the bearing of diapause 
induction on the phenology can perhaps be more easily appre- 
ciated. The function here is to forestall the onset of adverse 
conditions. The following are some examples involving photo- 
periodic control. 

The life cycle of the red spider mite Panouyclus ulmi is 
typical of species with several annual generations. The long 
daylengths of summer are responsible for the appearance of 
several successive generations of 'summer' females laying non- 
diapause eggs on the leaves. Under English conditions females 
laying winter (diapause) eggs appear in the penultimate and 
last (4th and 5th) generations in response to the reduced day- 
lengths of September. These individuals normally deposit their 
eggs on the bark of the tree long before leaf fall. However, 
heavy damage to the foliage by large mite populations often 
causes premature leaf abcission. It is interesting to find that 
under such conditions of semi-starvation diapause is also 
induced prematurely, even if long day conditions are still 
prevailing. In this respect the response to nutrition appears as 
a kind of 'double assurance'. 

Since day length provides a fixed 'point of reference', it might 
be expected that the composition of the terminal generations, 
and even the total number of generations in a season, would 
depend on the environmental temperature — a much more 
variable component. This has indeed been demonstrated in the 
moth Polychrosis by Komarova^^. Although this species is 
always bivoltine in the southern parts of the USSR, the inci- 
dence of diapause in the second generation varies from year to 
year and from place to place. In a cool year or at high altitudes 
the second generation is delayed relative to the critical photo- 
period with the result that a much higher proportion of indivi- 
duals enter diapause. 

I have already mentioned that some short day insects have 
been described. The function of this response may be simply to 
invert the season of dormancy. In the leafhopper Stejwcranus 
for example, a long photoperiod induces a summer (aestivating) 



INSECT DIAPAUSE AND ENVIRONMENT 127 

diapause. Bombyx, however, has a normal hibernation and the 
reversed response stems from a different cause. This is connected 
with the early occurrence of the sensitive Qgg stage in spring. 
A reversed response is therefore necessary to prevent diapause 
from supervening after one generation. In species such as 
Panonychus this difficulty does not arise since the sensitive 
stage occurs much later in the life cycle; and in addition, 
dormancy is maintained until the short day spring period has 
been almost completed. 

More complicated relationships are also known. According 
to Masaki^^ the Japanese magpie moth Abraxas miranda 
exhibits two separate types of pupal diapause which differ in 
intensity. The more intense summer diapause is induced by a 
moderately long photoperiod of 14-16 h, while the weaker 
diapause is induced in early winter by a somewhat shorter 
photoperiod of 11-13 h. Like other 'short day' insects, a day 
length of 7-9 h induces development without diapause. This 
response has the effect of imposing a bivoltine pattern on the 
annual cycle. Larvae pupating early in winter have a short 
dormancy whilst those doing so later on (they feed on ever- 
green Euonynms) develop without interruption. The apparent 
function of this adaptation is to synchronize the emergence of 
the moths in spring. 

GEOGRAPHICAL VARIATIONS 

Since the control of diapause involves rather precise relation- 
ships with photoperiod and temperature, it is not surprising to 
find that the character of the diapause in widely distributed 
species is by no means uniform, and that the differences are 
determined genetically. 

Diapause races are sometimes surprisingly local in their 
distribution. For example, there are at least three strains or 
races of Locusta migratoria in southern France alone-^. How- 
ever, other species show continuous transitions in the environ- 
mental response. Thus Danilyevsky'^i has found that the 

References p. 128 



128 A. D. LEES 

critical photoperiod is smoothly graded in populations of the 
moth Acronycta rumicis and ranges from nearly 20 h of light 
daily in the Leningrad populations to about 14i h in populations 
from the Black Sea coast. In the South moderate or high 
temperatures are also more effective in preventing diapause- 
induction by short days. The tendency in nature is therefore for 
the period of active growth to extend into the winter season and 
even to continue through it. Very similar relationships have 
been demonstrated in the red mite Tetranychus urticae'^'^ and in 
Anopheles maculipennis-^. 

The genetic examination of this interesting material is in its 
early stages. But it has already been shown in Acronycta that 
Fi hybrids of local photoperiodic races respond to intermediate 
critical photoperiods. And no clear-cut segregation emerges 
when the Fi progeny is backcrossed with the original forms-^. 
The type of inheritance is therefore polygenic. 

These results immediately indicate the high adaptive value 
which must attach to the correct adjustment of the diapause- 
inducing and diapause-terminating mechanisms. They also 
suggest that there must be considerable selective advantage in 
retaining the plasticity and variability of the diapause character. 

REFERENCES 

1 D. Keilin, Proc. Roy. Soc. (London), B, J 50 (1959) 149. 

- H. E. HiNTON, Proc. Zool. Soc. (London), 121 (1951) 37. 

3 H. E. HiNTON, Proc. Roy. Entomol. Soc. (London), (C), 25 (1960) 7. 

■1 A. S. Danilyevsky, Doklady Akad. Nauk S.S.S.R., 60 (1948) 481. 

5 A. D. Lees, Ann. Appl. Biol., 40 (1953) 449. 

« M. J. NoRRis, Nature, 181 (1958) 58. 

' H. J. MiJLLER, Zool. Anz., 760 (1958) 294. 

8 R. D. Hughes, /. E.xptl. Biol., i7 (1960) 218. 

« A. D. Lees, Ann. Appl. Biol., 40 (1953) 487. 
^° O. H. Paris and C. E. Jenner, in Photoperiodism and Related Phenomena 

in Plants and Animals, (ed. Withrow), A.A.A.S., Washington, 1959. 
^1 A. D. Lees, The Physiology of Diapause in Arthropods, Cambridge Uni- 
versity Press, London, 1955. 
1- R. C. Dickson, Ann. Entomol. Soc. Am., 42 (1949) 511. 
^'•^ (IvANCiCH) P. Gambaro, Arch. zool. itai, 42 (1957) 511. 



INSECT DIAPAUSE AND ENVIRONMENT 129 

1^ H. Tsuji, Japan. J. Appl. Entomol. Zool., 3 (1959) 34. 

15 H. A. SCHNEIDERMAN AND J. HORWITZ, /. Exptl. BioL, 35 (1958) 520. 

16 R. Perron, Acta Zool. (Stockholm), 35 (1954) 71. 

1' I. W. KozHANTSHiKOv, Eutoiuol. Obozrcniye, 31 (1950) 178. 

18 O. S. KoMAROVA, Doklady Akad. Nauk S.S.S.R., 68 (1949) 789. 

19 S. Masaki, Japan. J. Appl. Entomol. Zool., 2 (1958) 285. 

20 J. R. Le Berre, These, Fac. Sci. Univ. Paris, 1957. 

21 A. S. Danilyevsky, Entomol. Obozreniye, 36 (1957) 5. 

22 N. V. BoNDARENKO AND K. Khai-Yuan, Doklady Akad. Nauk S.S.S.R., 
119 (1958) 1247. 

23 N. K. Shipitsina, Med. Parasitol. and Parasitol. Bull., Moscow, 1 
(1959)4. 

2^ A. S. Danilyevsky, Vestnik Leningrad Univ., 21 (1957) 93. 

DISCUSSION 

Galun: Since insects do not see red light, it would be 
interesting to compare the spectrum for photoperiod sensitivity 
with the light spectrum. 

Lees: There is very little information on this subject although 
Geispits has examined various species of Lepidoptera from this 
point of view. He found that roughly the same wavelengths 
produced visual and photoperiodic responses and he therefore 
concluded that the simple eyes were in fact the photoperiodic 
receptors. But even if the action spectrum is identical this 
conclusion may not be warranted. 

Galun: It could indicate that the same pigment is involved, 
if not the same organ. 

Lees: Yes. Photolabile pterins have recently been demon- 
strated in the insect compound eye; and it is possible that we 
should think of these substances in searching for the photo- 
periodic pigment. 

Kohn: You said that diapause is an inherited characteristic 
rather than a condition, but you mentioned too that environ- 
mental influences on the adult insect resulted in changes in the 
diapause, the larvae and the eggs. You also said that there must 
be a way of transferring the information from the adult to the 
stage in which diapause occurs. Would it be possible that the 
information is recorded on genetic material? 



130 A. D. LEES 

Lees: I do not think that genetic material is involved. In the 
silkworm Bombyx the late embryos are light-sensitive and the 
effect is finally seen in the adult moth. Thus although a long 
time interval is involved, it is still the same individual and no 
inherited effect need be invoked. In Mormoniella the photo- 
periodic experience of the mother is transmitted to her fully 
grown larval offspring. But in both cases the effect is confined 
to one generation. In the next generation the switching mecha- 
nism can again be operated in either direction by the appro- 
priate environmental conditions. 

Nachmony: Is photoperiod only effective in inducing diapause 
or also in breaking it? 

Lees: In some cases it is effective in both. One example is the 
leafhopper Stenocranus which aestivates in summer under long 
day conditions but emerges from diapause in autumn in 
response to shorter daylengths. A second generation is then 
produced. The moth Dendrolimiis becomes dormant as a larva 
in autumn and then feeds and grows very little until the day- 
length has lengthened beyond the critical photoperiod in spring. 
Nevertheless, such examples are scarce. The diapause-inducing 
and diapause-terminating factors are usually quite different. In 
most species it is presumably an adaptive advantage to have 
separate timing mechanisms governing these two events, since 
it is unlikely that the optimal dates for diapause induction and 
termination will have the same daylength. 

Hestrin: Could you tell us more about the fly Polypedilum 
you mentioned? How was its remarkable resistance cO desicca- 
tion discovered? 

Lees: It was first collected by F. L. Vanderplank from 
shallow rock pools near Kaduna in Nigeria. During the rainy 
season these pools are full of water and the larvae can be 
readily observed. During the dry months the pools contain 
nothing but a small cake of dried mud. Most entomologists, I 
think, would pause to wonder what had happened to the 
insects. This problem was solved when some mud was soaked in 



INSECT DIAPAUSE AND ENVIRONMENT 131 

water. All the experimental work on this species has been 
carried out by H. E. Hinton at Bristol. 

Hestrin: Was any attempt made to grow this fly in the 
laboratory? 

Lees: Hinton has attempted it. but it has proved difficult. 

Galun: Long day and low temperature induce diapause, 
while in short days the high temperature induces it. What 
happens if you artificially reverse one of the conditions, so that 
you induce with one element and defer with the other. 

Lees: It works additively. If a long day insec ;is given a short 
day with high temperature, the latter will prevent at least some 
of the individuals from entering diapause. This lack of tempera- 
ture com_pensation may be adaptively useful, since rapidly 
breeding species can complete an additional generation under 
short dav conditions. 



THE ENDOCRINOLOGY AND BIOCHEMISTRY OF 

INSECT DIAPAUSE 

A. D. LEES 

Agricultural Research Council, Unit of Insect Physiology, 
Cambridge (Great Britain) 

The blood-sucking reduviid bug Rhodnius invariably under- 
goes ecdysis after taking a full blood meal. In 1934Wigglesworth^ 
observed that insects decapitated immediately after the meai 
failed to moult, although they often remained alive for over a 
year. He concluded that they had been deprived of a growth 
hormone produced in the head. His comparison of this experi- 
mentally induced state with other naturally occurring examples 
of dormancy has since been shown to have a general validity. 

As knowledge of the humoral control of moulting and meta- 
morphosis increased, it became apparent that the source of the 
moult-inducing hormone was the brain or, more precisely, the 
gi'oups of neurosecretory cells located near the mid-line in the 
dorsum of the forebrain. The axons of these cells were shown to 
end in two small organs situated just behind the brain, the 
corpora cardiaca. Droplets of secretion, presumably transporting 
the hormone, pass down the axons to the cardiaca and are there 
discharged into the blood. 

This relatively simple picture was considerably complicated 
when it was shown that a further endocrine organ — the pro- 
thoracic glands — also secreted a moult-inducing hormone. 

The apparent contradiction was resolved by WiUiams- who 
showed that the neurohormone from the brain activates the 
prothoracic glands. It is the hormone secreted by the latter 
organs — ecdyson — which stimulates the tissues to undergo a 
moulting cycle. Since larval and pupal diapause involves the 
suspension of ecdysis and metamorphosis it is not surprising 
that the same dual endocrine mechanism controls diapause. 
Indeed, the material used by Williams for demonstrating the 
endocrinological relationships of the brain and prothoracic 



ENDOCRINOLOGY OF INSECT DIAPAUSE 133 

glands was a diapausing insect, the giant silkmoth Hyalophora 
cecropia. The combination of large size and tolerance of drastic 
surgical procedures makes this the material of choice. 

When WiUiams removed the brains from diapausing cecropia 
pupae he found that they failed to develop even if chilled for 
the length of time which would normally bring dormancy to an 
end. On the other hand, when brains from chilled pupae were 
transplanted into unchilled pupae, development was promptly 
initiated. The interaction of brain and prothoracic glands was 
tested by implanting these organs, either separately or together, 
into the isolated pupal abdomen, which is without any known 
endocrine source of its own. Moulting and metamorphosis was 
only caused when both organs were present. 

These results suggested that the immediate cause of diapause 
in the cecropia pupa was the inactivity of the neurosecretory 
brain cells, and that the failure of growth was due to the 
absence of a promotive hormone (ecdyson) rather than to the 
presence of an inhibitory factor. This hypothesis was tested by 
grafting together a chilled and an unchilled pupa. Both members 
of the pair developed, indicating that no inhibitory substances 
of any significance were present in the unchilled insect. This 
conclusion appears to hold good in many species. Indeed, 
growth inhibitors of the type found in plant seeds are unknown 
in dormant insects. 

As might be expected, the brain-prothoracic gland system has 
also proved to be the main controlling system in larval diapause. 
Nevertheless, it may well be that a further endocrine — the 
corpus allatum — is sometimes involved. It is well known that 
in larval insects this organ produces a secretion — the juvenile 
hormone — which leads to the retention of the larval characteris- 
tics. Unmistakable signs of activity have been found in the 
corpora allata of some diapausing insects, for example in the 
larvae of the rice stem borer Chilo^. However, it is still uncertain 
whether these organs play any positive role in stabilizing 
diapause. 

The humoral control of embryonic diapause, which has been 

References p. 140 



134 A. D. LEES 

studied mainly in the silkworm Bombyx, involves a different 
combination of endocrine organs, a particularly important 
centre being the suboesophageal ganglion with its associated 
neurosecretory cells. Pupae and moths, determined by their 
previous photoperiodic treatment to be non-diapause egg- 
producers, can readily be induced to lay diapause eggs by 
implanting ganglia from diapause egg-producers^. The release 
of the 'diapause hormone' from the suboesophageal ganghon is 
considerably modified by the brain, which in this instance 
apparently acts through the nervous paths in the circum- 
oesophageal commissures. It can either promote or inhibit the 
synthesis and release of the hormone in accordance with the 
previous photoperiodic treatment^. The corpus allatum also 
seems to play some part in diapause regulation, its effect being 
antagonistic to that of the suboesophageal ganglion. But here 
again a general control of its activity seems to be exercised by 
the brain^. 

Suboesophageal ganglia that are active in causing Bombyx to 
lay diapause eggs have also been obtained from certain species 
with a pupal diapause. Since the 'diapause hormone' is not 
known to be concerned in controlling pupal diapause, it seems 
likely that this secretion is utilized for other physiological 
functions. In this context it is worth recaUins that the cockroach 
suboesophageal ganglion secretes the neurohormone responsible 
for initiating and maintaining the diurnal rhythm of activity^. It 
remains to be decided whether these substances have anything 
in common. 

Although the maternal endocrine systems which control the 
production of the two egg types in Bombyx have been the 
subject of detailed examination, the actual mechanism which 
leads to the growth failure in the embryo is still obscure. There 
is no reason to suppose that the 'diapause hormone' from the 
suboesophageal ganglion is passed into the egg and acts as an 
inhibitor. When diapausing embryos of Bombyx are dissected 
out of the tgg and are suspended in a hanging drop side by side 
with a non-diapause embryo, the growth of the former is 



ENDOCRINOLOGY OF INSECT DIAPAUSE 135 

Stimulated^. This seems to indicate that some substance required 
for embryonic growth is lacking in the dormant embryo. 

In most locusts and grasshoppers embryonic growth cannot be 
completed until water is taken up from the environment. Slifer^ 
has shown that diapause can be terminated in the Qgg of 
Melanoplus dijferentialis if a waxy layer is removed from the 
surface of the hydropyle — the special water-absorbing area of 
the egg-shell. The effective solvents include medicinal paraffin 
which is so innocuous to living tissues that any stimulatory 
effect through 'wounding' is extremely improbable. Yet the 
causal mechanisms which control the ordered appearance and 
disappearance of such physical and physiological barriers are 
still unknown. The mode of action of low temperature in 
bringing about the final dissolution of the wax layer is one such 
problem. Unlike the egg of Bombyx, which does not take up 
water, control does not seem to reside in the embryo itself, for 
Bucklin^^ has found that diapausing embryos, when 'explanted' 
in a hanging drop of Ringer, immediately resume their develop- 
ment. 

Although the corpus allatum ceases to secrete juvenile hor- 
mone just prior to metamorphosis, it often displays renewed 
activity during adult life. Indeed, its secretion at this time has 
been shown to be necessary for the maturation of the eggs of 
many insects. Since adult diapause is characterised by repro- 
ductive inactivity, it was suspected that the corpus allatum might 
be involved. This view has been proved correct by De Wilde^i 
who showed that Colorado beetles (Leptinotcusa) that are 
about to enter diapause in response to a short photoperiod can 
be induced to leave the soil, reverse their geotaxis, feed and lay 
eggs, by implanting active corpora allata. The corpora a/ lata of 
this insect m.ay also be influenced by the brain. 

MODE OF ACTION OF ENVIRONMENTAL FACTORS 

In my first lecture I gave some account of the way in which 
environment controlled diapause induction and diapause termi- 

References p. 140 



136 A. D. LEES 

nation, but hardly touched on the physiological mechanisms 
which must be involved. 

We have seen that diapause is frequently evoked by the 
appropriate photoperiod, temperature or nutrition. It has been 
shown that blinding fails to eliminate the photoresponse. 
Neither the stemmata (simple larval eyes) nor the compound 
eyes are therefore concerned. The photoreceptors have not yet 
been identified in a diapausing insect. Nevertheless, it is certain 
that in aphids with a similar photoperiodic reaction (which, 
however, governs a different process — the formation of virgino- 
parous and oviparous offspring) the receptor lies inside the 
animal. When different areas on the head, thorax and abdomen 
were illuminated with small spots of light for the necessary time, 
leaving the rest of the body dark, a positive response could only 
be elicited from the dorsum of the brain (Lees, unpublished 
results). Whether the photosensitivity of this region is due to the 
presence of neurosecretory cells or two other light-sensitive 
structures (perhaps neurones), remains to be decided. 

Little is known about the role of temperature ; but a clue to 
the possible mode of action of nutrition on diapause induction 
is provided by the work of Johansson^^ ^j^q showed that the 
failure of Oncopeltus to mature eggs when starving is due to the 
inhibitory action of the brain on the corpus allatum; this 
influence disappears when the insect is fed or the allatal nerve 
cut. A comparable relationship in which nutrition exerts a 
controlling influence on the activity of the brain may exist in 
diapausing insects. 

This information, scanty though it is, suggests that the brain 
may be the centre on which diapause-inducing stimuli act. 
Certainly, the role of the brain in terminating diapause has been 
proved conclusively. You will recall that unchilled cecropia 
pupae end their diapause when supplied with a brain from a 
chilled pupa. Davis and Schneiderman (unpublished results) 
have demonstrated that this is indeed the site of action of low 
temperature since brains chilled in vitro at 6° in a medium of 
haemolymph for 8-14 weeks become competent to end diapause. 



ENDOCRINOLOGY OF INSECT DIAPAUSE 137 

Van der Klooti-^ has shown that the humoral inactivity of the 
brain during diapause is accompanied by a fall in the level of 
cholinergic substances. The progress of chilling is marked by a 
gradual rise in the titre of acetyl choline and finally by the 
reappearance of cholinesterase and the resumption of electrical 
activity. The virtual depolarisation of the brain neurones during 
diapause appears to serve the purpose of 'protecting' the 
neurosecretory cells either from possible sensory stimulation or 
from random discharges from other neurones. 

Although the diapause-completing processes which take place 
in response to chilling may be associated with changes in the 
ordinary neural tissues of the brain, it may well be that the 
primary events take place within the specialised neurosecretory 
cells. If so, these processes must be extremely complex, as the 
following well analysed instance illustrates. Schneiderman and 
Horwitz^^ have shown that diapause in the hymenopterous 
parasite MormonieJla can be terminated by chilling the larvae 
at 5° for an adequate period. But if the chilling is interrupted by 
periods of warming, the effect of low temperature is largely 
undone. The chilling process is therefore reversible. A further 
complication is that the temperature characteristics of the 
diapause-completing process undoubtedly change with time. 
The first stage requires moderate warmth, although diapause 
can never be ended in these conditions. The second stage 
requires low and the final stage moderately high temperatures. 
Different phases in diapause completion can also be separated 
by studying the effects of oxygen lack. It turns out that the 
chilling process is aerobic, whilst the final high temperature 
phase is favoured by anoxia. 

The action of low temperature can perhaps be most simply 
seen in terms of competing reactions with different temperature 
coefficients. The model proposed by Schneiderman and Horwitz 
consists of a synthetic reaction opposed by an oxidative break- 
down process. Low temperature slows down the latter reaction 
but not the synthesis, thus permitting the brain to accumulate 
the substance necessary for the production of the neurohormone. 

References p. 140 



138 A. D. LEES 

Recent observations by Way^^' ^^ on the fly Leptohylemyia 
are particularly interesting in this connection. The eggs of this 
species exhibit two optima for diapause termination, at approxi- 
mating +2° and — 20°. As the latter temperature is probably 
incompatible with biochemical activity, Way has suggested that 
the physical rupture of a lipoprotein membrane — perhaps even 
some element of the neurosecretory cells — may be involved. 

Histological methods have as yet contributed rather little 
towards unravelling these events. In some species, however, 
promising results have been achieved. The hawk moth Mimas is 
a case in point. There it has been possible to correlate the 
chilling process with the synthesis of neurosecretory material 
and to trace its passage down the axons to the corpus cardiacum^^' . 

RESPIRATION AND METABOLISM 

Diapausing insects are characterized by a low level of metab- 
olism. In the cecropia silkworm., for example, a precipitous fall 
in respiration takes place as the larva pupates. Throughout the 
months of diapause oxygen consumption continues at a very low 
level, only to increase dramatically as the prothoracic gland 
hormone is secreted^^. 

Bodine and his co-workers many years ago drew attention to 
the possibility that diapause is associated with some change in 
the respiratory enzymes, particularly the cytochrome system. 
This was indicated by the insensitivity of their material — the egg 
of Melanoplus — to cyanide. This view has since been confirmed 
and greatly extended by Williams and his associates who showed 
that the low diapause metabolism of the cecropia moth is also 
unaffected by cyanide and carbon monoxide. Since these in- 
hibitors combine with cytochrome c oxidase, this result was 
thought at one time to indicate that electron transfer proceeded 
by a diff'erent pathway, the terminal oxidase being a flavoprotein 
or perhaps an auto-oxidisable cytochrome b. 

Spectroscopic studies of the cytochromes in the pupal tissues 
have shown that cytochrome b-^ and cytochrome oxidase are 



ENDOCRINOLOGY OF INSECT DIAPAUSE 139 

indeed present during diapause, although in somewhat reduced 
amounts. But the most sisnificant change is the virtual dis- 
appearance of cytochrome r^^. 

The action of dinitrophenol on the diapause respiration is 
important in this context. Dinitrophenol is known to increase 
the turnover of respiratory carriers, probably by uncoupling 
oxidative phosphorylation from electron transfer, and so in- 
creasing the demand for oxygen. In the cecropia pupa dinitro- 
phenol injections may cause a seventeen-fold rise in oxygen 
uptake. Moreover, the dinitrophenol-stimulated respiration is 
CO-sensitive. showing that dinitrophenol increases the satura- 
tion of cytochrome oxidase-^. 

These results have led to the suggestion that CO- and CN- 
insensitivity arises as a result of the great excess of cytochrome 
oxidase in the diapausing tissues relative to cytochrome c. A 
high proportion of the enzyme is therefore unsaturated and 
can be immobilized with inhibitors without affecting electron 
transfer from cytochrome c. Thus cytochrome oxidase may well 
be the terminal oxidase in diapausing as well as in growing 
insects. 

The cecropia pupa is a rather extreme example of the insensi- 
tivity often shown by dormant insects to respiratory inhibitors. 
Other species are much less resistant — the weevil Sitona. which 
hibernates as an adult, is an example-^. Such differences are 
probably related to the amount of muscle present in the dia- 
pausing insect. In the cecropia pupa there is little of this tissue 
save for the small intersegmental muscles which serve to flex 
the abdominal segments. These muscles do in fact retain an 
intact cytochrome system, but their total volume is small relative 
to the pupa as a whole. Dormant larvae or adult insects are 
more mobile and retain a correspondingly large proportion of 
muscles. These of course contain the sarcosomes with which the 
cytochrom_e system is associated. 

A very interesting finding which is still incompletely under- 
stood concerns the striking stimulation of respiration which 
takes place when a diapausing insect (for example, the cecropia 

References p. 140 



140 A. D. LEES 

pupa) is wounded or subjected to surgical operations. The 
effect is not localised but is due to increased oxygen consump- 
tion throughout the body. The stimulated respiration is certainly 
associated with the synthesis of cytochrome c, since the stimu- 
lated respiration is inhibited by CO. Nevertheless, it is significant 
that although the metabolism may be raised almost to the level 
found in the developing insect, no development in fact takes 
place, since there is no secretion from the prothoracic glands. 
The endocrine system is therefore the prime mover in the 
control of diapause. 

REFERENCES 

^ V. B. WiGGLESW'ORTH. Quart. J. Microscop. Sci., 77 (1934) 191. 

2 C. M. Williams, Biol. Bull., 93 (1947) 89. 

3 M, FuKAYA AND J. MiTSUHASHi, Japan. J. Appl. Entomol. ZooL, 2 (1958) 
223. 

■^ K. C. Hasegawa, J. Fac. Agr. Tottori Univ., 1 (1952) 83. 

5 S. FuKUDA, Ann. Zool. Jap., 25 (1952) 149. 

6 S. MoROHOSHi, /. Inst. Physiol, 3 (1959) 28. 

7 J. E. Marker, Nature, 173 (1954) 689. 

8 T. Takami, Science, 130 (1959) 98. 

» E. H. Slifer, /. Exptl. ZooL, 138 (1958) 259. 

1° D. H. BucKLiN, in Physiology of Insect Development, University of 
Chicago Press, Chicago, 111., 1959. 

11 J. de Wilde, Arch, neerl. zool, 10 (1954) 375. 

12 A. S. Johansson, Nature, 181 (1958) 198. 

13 W. G. VAN DER Kloot, Biol Bull, 109 (1955) 276. 

1^ H. A. ScHNEiDERMAN AND J. HoRWiTZ, /. Exptl Biol, 35 (1958) 520. 

15 M. J. Way, Trans. Roy. Entomol. Soc, HI (1959) 351. 

i« M. J. Way, J. Inst. Physiol, 4 (1960) 92. 

1" K. C. HiGHNAM, Quart. J. Microscop. Sci., 99 (1958) 73. 

18 H. A. SCHNEIDERMAN AND C. M. WiLLIAMS, Biol Bull, 105 (1953) 320. 

1^ D. G. Shappirio and C. M. Williams, Proc. Roy Soc. (London) B., 147 
(1957) 233. 

20 C. G. Kurland and H. A. Schneiderman, Biol. Bull, 116 (1959) 136. 

21 K. G. Davey, Can. J. Zool, 34 (1956) 86. 



ENDOCRINOLOGY OF INSECT DIAPAUSE 14] 

DISCUSSION 

Galun: Does Carrol Williams' experiment mean that H. 
cecropia does not need any gonadotropic hormone for egg 
maturation? 

Lees: That is so. The control of egg development by hormone 
varies greatly in different insects. Ccilliphora requires material 
from the neurosecretory brain cells for egg development. Many 
require juvenile hormone from the corpus aUatiim, but H. 
cecropia pupae will develop viable eggs after removal of the 
corpora allata. The puzzling thing is that the corpora allata of 
male H. cecropia adults produce large quantities of hormone 
which the moth does not appear to require at all. 

Galun: Is it possible, in your experiment with aphids, to 
illuminate the internal organs directly rather than through the 
epidermis, and get the same effect? 

Lees: It would be very difficult technically, since the aphids 
must be allowed to feed freely on the host plant after each cycle 
of illumination. 

Pener: What is your opinion on Bodenstein's experiment on 
the disappearance of the thoracic gland after the last moult in 
the adult cockroach? But when the corpora allata had been 
extirpated the thoracic gland remained intact and extra moulting 
of the adult occurred. 

Lees: As in other insects, attainment of the adult state in 
Periplaneta is accompanied by the degeneration of the thoracic 
glands; and this is the immediate reason for the cessation of 
moulting. Bodenstein has shown that, under experimental 
conditions, the thoracic glands can be caused to remain active 
in the adult, and the adult then moults. The necessary conditions 
for preserving the thoracic glands seem to be satisfied when the 
corpus cardiacum is active and the corpus allatum inactive. 

Shulov: I should like to refer to an experiment made on 
larvae of Trogoderma granaria. One set of larvae continued to 
moult over a period of eight months becoming gradually smaller 
until they became microscopic. The other set did not moult at 



142 A. D. LEES 

all. Yet both sets came from the same breed and no differences 
in external conditions seemed to exist. 

Lees: I can think of no convincing explanation for the type 
of variation you mention. But I would like to call attention to 
the recent work of Burges on the same species. Larvae, which 
from their low respiratory rate would be judged to be in dia- 
pause, nevertheless still moult at intervals. In the process they 
often become smaller. 

Reinhold : Is there not a parallelism between the waking from 
diapause and the stimulation or fertilisation of sea-urchin eggs? 
The unfertihsed eggs, for example, have a respiration not 
sensitive to cyanide, and become sensitive to cyanide on fertili- 
sation. 

Lees: Yes, I think you could call the unfertihsed egg a dia- 
pausing egg. 

Muehsam: You mentioned a case of the influence of neuro- 
secretory brain cells on oviposition, where the cutting of the 
axons resulted in oviposition in an unfed adult. On which 
insect was this experiment done? 

Lees: On Oncopeltus. Johansson has shown that in the fasting 
insect the brain inhibits the corpus allatum through the com- 
missures. When these are cut, the inhibition ceases and egg- 
laying begins. There is, of course, much variation according to 
species. For example. Ellen Thomsen has shown that quite a 
different mechanism is in control of egg maturation in Calliphora. 

Harpaz: I would like to draw an analogy becween the 
breaking of diapause in insects and dormancy in seeds, referring 
to the mechanism of mechanical injury. There is evidence from 
Theron that the diapausing codling moth larvae in a cocoon 
repair the cocoon even if the latter is torn several times, but 
finally the insect gives up. pupates and resumes its development. 
This could provide an analogy to mechanical injury in seeds. 

Lees: These experiments were repeated by Andrewartha in 
Cambridge who did not succeed in breaking diapause by 
removing the cocoon. This was, of course, an English strain not 
a South African one, as in Theron's case. 



ENDOCRINOLOGY OF INSECT DIAPAUSE 143 

Harpaz: In experiments we have conducted, diapause was 
terminated by cold shock treatment, consisting of chilling to 
— 10° followed by immediate transfer to 25°. This procedure 
probably has no ecological equivalent, as nowhere are such 
conditions extant. 

Lees: I think I must disagree with you here. There are some 
investigators who believe that this finding does have an ecologi- 
cal application. These include Danilyevsky who found that in 
the moth Satumia pavonia very low temperatures can terminate 
diapause; and Way has made similar observations on the fly 
Leptohylemyia. These authors point out that in continental 
regions of the north temperate zone, the warmest part of the 
winter comes first, really cold conditions being delayed until the 
early months of the year. It is possible that this final stimulus is 
required to complete the processes which result in the release 
from diapause. 

Hestrin: In view of the long time period which elapses until 
the effect of temperature change on the eggs becomes manifest, 
one might wonder whether the effect might not involve crystalli- 
sation or solubilisation of components present in the cytoplasm. 
Has it been possible to see alterations by microscopic examina- 
tions of the tissues? 

Lees: Yes, cytological changes in the neurosecretory cells of 
a few diapausing insects have been described, but their signi- 
ficance is doubtful. So far, such changes have merely been taken 
as indicating that the particular cells are actually involved in 
diapause control. 



ENVIRONMENTAL FACTORS IN INTERRUPTION 
OF DEVELOPMENT OF ACRIDIDAE EGGS 

A. S. SHULOV AND M. P. PENER 

Laboratory for Entomology and Venomous Animals, 

Department of Zoology, Hebrew University, 

Jerusalem (Israel) 

The ways in which Acrididae eggs may develop are manifold. 
The eggs of tropical and subtropical locusts Schistocerca 
gregaria (Forskal) and Lociista migratoria migratorioides (R. and 
F.) usually develop continuously and hatch within a compara- 
tively short period. Some others also develop continuously under 
field conditions, but there may be a slow development at lower 
temperatures, as in the case of Pareuprepocnemis syriacus 
(Table I). 

There are many other types of development which include 
interruption at various stages of the embryonic development, 
such interruptions being caused by intrinsic as well as extrinsic 
factors, e.g. temperature and humidity or both these factors 
combined. 

Slifer^, Steele', Bodenheimer and Shulov^ and Shulov and 
Pener^ provided the basis for the study of embryonic develop- 
ment in some Acrididae by describing morphological stages in 
connection with the time of their appearance (Fig. 1). 

Shulov^ classified interruptions in embi*yonic development of 
Acrididae according to four types. 

The first may occur in the initial period after oviposition, 
when the Qgg is dormant and no embryonic germ-band appears 
during a period of two to four weeks. This type of pause appears 
in Dociostaurus maroccanus^, Austroicetes cruciata'^ and in 
Tmethis pulchripennis asiaticus^. In Dociostaurus, the eggs during 
this period are not influenced by temperature. The water content 
of the eggs of Dociostaurus and Tmethis is at equilibrium or they 
lose some water: the addition of water during this period kills 
the eggs of both species. 



INTERRUPTED GROWTH OF ACRIDIDAE EGGS 



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146 



A. S. SHULOV AND M. P. PENER 



The second type includes those eggs which show a slow 
development during anatrepsis, iniluenced to a certain degree 
by rise in temperature, as in the cases of Locusta migratoria"' , 
Tmethis pulchripennis asiaticus^ and Calliptamus palaestinensis^. 
In Dociostaurus^ the influence of temperature at this stage is not 
significant and this period may last up to five months. 

The eggs during this period are not affected by addition of 
water in Calliptamus and Locusta, but are spoiled in the case of 
Dociostaurus and Tmethis. 

The third type occurs at the end of anatrepsis and includes all 
forms of diapause which cannot be broken by changes of 
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INTERRUPTED GROWTH OF ACRIDIDAE EGGS 



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different scales, from the ventral side. Lateral views of some of the stages 

are also included. 



References p. 151 



148 



A. S. SHULOV AND M. P. PENER 




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Fig. 2. Embryos of Locusta migrator ia migratorioides (R. and F.) (stages 
XIV, XVIII, XIX) and of Dociostaiirus maroccanus Thnb. (stage XIV, in 
the right corner (punctated)). {Locusta embryos according to Shulov and 
Pener-*, Dociostaurus embryo according to Bodenheimer and Shulov^^). 
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rus maroccanus shown. The thoracal appendages of the Dociostaurus embryo 
show more advanced differentiation, in comparison to the Locusta embryo 

of the same stage. 



ready morphologically for katatrepsis, but not physiologically. 
This type of interruption is known in Dociostaurus^ and in 
diapause-bound eggs oi Locustana pardalina^. The diapause may 
last from two to several weeks. It seems that in Dociostaurus 
this period is not influenced by temperature or humidity, but in 
Locustana the addition of water at this time has apparently an 
adverse eff"ect, i.e. causes prolongation of the diapause. 

The fourth type of interruption comprises embryos at the end 
of anatrepsis which will resume development if provided with 
a sufficient amount of water. The interruption in such cases may 
persist for various lengths of time, ranging from five months in 
the eggs of Calliptamus paJaestinensis^ to more than three years 
for the eggs of Locustana pardalina'^^ (Table II). 

The species m_entioned in Table II (part A) are typical for 
arid climates. Their eggs are laid mostly in dry soil. Such species 



INTERRUPTED GROWTH OF ACRIDIDAE EGGS 



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References p. 151 



150 A. S. SHULOV AND M. P. PENER 

achieve a morphologically more advanced state at stage XIV, 
than species which do not as a rule show any diapause. We have 
been able to demonstrate that in Schistocerca gregan'a^^ as well 
as in Locusta migratoria migratorioides and Nomadacris sep- 
temfasciata (Shulov and Pener, manuscript in preparation), eggs 
deprived of the full quantity of water needed for completing 
development reach stage XIV only, and then enter into a develop- 
mental pause which may be broken in the same way as in the 
above group, i.e. by addition of water (see Table II, part B). 
However, the morphological stage in which this interruption 
occurs is significantly less differentiated than is the stage XIV of 
eggs which interrupt their development at the fourth type of 
diapause almost always under natural conditions (Table IIA)^. 

Two further types of interruption may be added to the four 
types already mentioned. 

One has been observed in Melanoplus dijferentialis, where the 
break of diapause is connected as a rule with low temperatures^ 
under natural conditions. This type of interruption of develop- 
ment occurs at the end of anatrepsis. Resumption of develop- 
ment generally takes place when the eggs are transferred from 
low temperature conditions to higher ones, but it is still possible 
that the upper limit of the lower temperature range responsible 
for the break of diapause merges with the lower limit of that 
temperature range which allows renewal of slow development 
after the end of the diapause. 

This type of interruption is manifested in the northern form 
of Locusta migratoria^'^, in CaUiptamus palaestinemis^ and 
possibly in Austroicetes cruciata^ and Chorthippus bnmneus^^. 
It is typical of temperate regions. 

A sixth type of interruption of development has been stressed 
by Slifer^^ regarding those Acrididae which enter a diapause 
shortly before hatching. This type of diapause appears in some 
species of the genus Melanoplus such as M. bivittatus and its 
duration seems to be shortened by the influence of low tempera- 
tures, near 5°^^. 

Another group seems to exist in which the development of 



INTERRUPTED GROWTH OF ACRIDIDAE EGGS 151 

eggs is interrupted by temperatures below their developmental 
thresholdi^. w^ ^re unable to classify them into the above 
proposed scheme, as the stage or stages at which the embryonic 
development is interrupted are not known to us. 

Apart from environmental factors such as temperature and 
humidity, it seems that the structure of the egg envelope, and 
especially that of the hydropyle, is of essential importance at 
least in Melanophis differeutiaJis type of eggs. Weathering and 
other changes in these structures play an important role in 
breaking of diapause, according to Slifer^'* and Lees^"^. 

The intrinsic factors connected with appearance and breaking 
of diapause in Acrididae are still unknown. It is quite possible, 
however, that their influence is not less important than that of 
the extrinsic influences. 

It is unnecessary to stress the survival eff'ect of diapause and 
its importance in the adaption of the species, cf. Acrididae, to 
their environment. We should like only to point out that, in the 
case of CaUiptamiis palaestinensis, in which interruptions in de- 
velopment of the fourth and fifth types combined take place, we 
have been able to show recently^ how well the way of the develop- 
ment fits the climatic conditions of the geographic region of its 
distribution. The eggs of CaUiptamus palaestinensis are laid in 
autumn and develop slowly in moist as well as in dry soil. For 
their successful development both cold temperatures of the 
local winter and the rains occurring late in spring, are essential. 

REFERENCES 

1 E. H. Slifer, /. MorphoL, 53 (1932) 1. 

•' H. V. Steele, Trans. Roy. Soc. S. Australia, 65 (1941) 329. 

^ F. S. BoDENHEiMER AND A. Shulov, Bull. Research Council Israel, J 

(1951) 59. 
-> A. Shulov and M. P. Pener, Locusta, 6 (1959) 73. 
'" A. Shulov, Proc. XIV Intern. Congr. Zool., Copenhagen 1953, No. 6, 

1956, p. 395. 
6 A. Shulov, Bull. Research Council Israel, 2 (1952) 249. 
■^ E. M. Shumakhov and L. A. Jakhimowitch, Zool. J., XXIX, 4 (1950) 

327. 



152 A. S. SHULOV AND M. P. PENER 

8 M. P. Pener and a. Shulov, Bull. Research Council Israel, 9 B (1960) 131. 

9 J. J. Matthee, Sci. Bull. Dept. Agr. S. Africa, No. 316 (1951) 1. 

10 J. C. Faure, Bull. Eutoinol. Research, 23 (1932) 293. 

11 A. Shulov and M. P. Pener, in the press. 

12 L. A. Jakhimovitch, Doklady Akad. Nauk S.S.S.R., 73 (1950) 1105. 

13 O. W. Richard and N. Waloff, Anti-Locust Bull., No. 17 (1954) 1. 

14 E. H. Slifer, J. Exptl. Zool., 138 (1958) 259. 

15 N. S. Church and R. W. Salt, Can. J. Zool., 30 (1952) 173. 

16 S. M. Zambin, Plant Protection, 19 (1939) 48. 

i'^ A. D. Lees, Cambridge Monographs in Experimental Biology, No. 4, 

Cambridge University Press, 1955. 
1^ A. G. Hamilton, Trans. Roy. Entoniol. Soc. London, 101 (1950) 1. 

DISCUSSION 

Lees: I would like to ask Dr. Shulov whether he thinks that 
Slifer's observation on the eggs of Melanoplus dijferentialis are 
applicable to his species. In Melanoplus Slifer demonstrated a 
physical barrier to the uptake of water, which is probably a 
waxy substance removable by mild solvents, such as medicinal 
paraffin. At the same time as the egg goes into diapause, this 
waxy layer is deposited on the hydropyle and development 
ceases. When the layer is dissolved and water absorbed, develop- 
ment continues. 

Shulov: We have been unable to find a similar mechanism 
regarding the formation of the hydropyle in Dociostaunis eggs, 
which under our conditions have a natural diapause. We find 
here another kind of grasshopper, Tmethys, which develops 
without any addition of water. Its hydropyle does not show any 
significant change during development. Therefore it appears to 
me that the physical change around the hydropyle is generally 
less important. 

Lees: Do you think that the low temperature acts upon the 
embryo or upon the cells underlying the hydropyle? 

Shulov: I could not say, since we failed to isolate viable 
embryos. 

Galun : Did you ever try to break the shell mechanically, in 
the case where the composition of the waterproofing material is 
unknown? 



INTERRUPTED GROWTH OF ACRIDIDAE EGGS 153 

Shulov: We tried it on Dociostaurus and found that pierced 
eggs imbibed water but the embryo did not develop and did 
not turn around. 

Pener : I would like to refer to my experiments on Calliptamus 
palaestinensis. The diapause eggs of this grasshopper were 
treated by Slifer's method, some of them with mineral (paraffin) 
oil and others with xylol, but development was not resumed. 
The eggs imbibed some water, however, a distinct imbibition 
was observed also in the untreated eggs during diapause. Some 
months later the treated and untreated eggs were transferred to 
low temperature and the majority resumed development. 



SOME OBSERVATIONS ON THE ROLE OF 
DIAPAUSE IN THE PHENOLOGY OF INSECTS IN 

SEMI-ARID ZONES 

I. HARPAZ 

Department of Entomology, Faculty of Agriculture, 
Hebrew University, Rehovot (Israel) 



A subtropical climate, in particular a Mediterranean climate, 
is characterized by a long dry summer and a mild rainy winter. 
Such an annual cycle induces in many insects a summer-heat 
and/or drought diapause, and in a smaller number of cases a 
winter-cold diapause. The latter is predominant in Central and 
Western Europe, and its manifold aspects have already been 
extensively studied there, so much so that hibernation and 
diapause are often used as synonyms. On the other hand, heat 
and drought quiescence of aestivating insects are phenomena 
which have been much less considered, particularly heat- 
diapause in its strictest sense. Incidentally, diapause, aestivation 
and quiescence are not wholly synonymous, as Lees^ has 
pointed out very clearly. Reference at this stage should be made 
to Rivnay's paper-, which was the first appraisal of the problem 
in this region. 

Let us take as an example the codling moth [Cydia pomonella 
L.). It is essentially univoltine in Northern France and England, 
where over 90 % of the fully-fed larvae enter diapause by the 
end of summer and pupate only the following spring. In the 
South of France, however, where the summer is warmer and 
development is faster, about 70 % of the larvae pupate before 
entering diapause and give rise to a second generation^. In 
Israel, with an even warmer summer, where the flight of the first 
generation takes place in April and May (as compared with 
June and July in Western Europe, including Southern France) 
there are even three generations a year^ with diapause setting in 
as from mid-July and onwards. This situation can be satis- 



DIAPAUSE AND INSECT PHENOLOGY 155 

factorily explained on the basis of Dickson's studies on the 
photoperiodicity of this moth, which conclusively demonstrated 
that the codUng moth responds to a critical day-length of 15 h, 
while with 12 h of daily illumination almost all larvae enter 
diapause^. It should be pointed out that in all the countries 
mentioned previously the codling moth caterpillars enter dia- 
pause as from mid-July at the earliest, i.e. at a period of 
shortening day-light hours. 

However, in the vicinity of Baghdad, where conditions would 
be expected to be favourable for an even greater number of 
generations than in Israel, it was observed by the late Prof. 
Bodenheimer that the larvae entered diapause as early as in the 
middle of May, and thus gave rise to no more than one annual 
generation. As diapause occurs there during a period of in- 
creasing day-length (the average number of day-light hours 
during May in Baghdad is 13 h 35 min), the diapause-inducing 
factor in Iraq should be sought elsewhere than in the photo- 
period effect. A simple experiment carried out in our laboratory 
at Rehovot^ revealed that larvae hatched during the first half 
of April, which after penetrating fruits were transferred to an 
incubator at 27°, all went into diapause, regardless of host fruit 
variety. The same thing happened when the larvae were exposed 
to 27° following some 17 days of natural outdoor development, 
which constitutes about three quarters of the total duration of 
larval development. On the other hand, when fully grown 
larvae were collected outdoors in May and exposed to the 
same 27° constant temperature, only 42% went into diapause 
and the rest pupated and emerged the same summer. Simul- 
taneously with this experiment, when another group of larvae, 
hatched in May, were exposed to a temperature of 23°, all 
pupated at the expected time. The average monthly tempera- 
tures in Baghdad^ are 22° in April, 28° in May and 32° in June, 
whereas in Rehovot the monthly mean temperature reaches 
26.5^ only in August, which is the time our codling moth begins 
its diapause. Another conclusion to be drawn from this experi- 
ment is that the stage of development sensitive to high tempera- 

References p. 157 



156 I. HARPAZ 

ture as a diapause-inducing factor is the last quarter of the 
duration of larval development, in other words the last instar. 

Undoubtedly diapause in the codling moth in general is 
governed by a variety of factors, i.e. photoperiod, temperature, 
diet and perhaps others yet unknown, each one of which may 
become predominant according to the prevailing circumstances. 

Perhaps a more striking example is the phenology of the 
onion fly (Hylemyia antiqiia Meigen) in Israel as recently 
reported by Yathom^. The same species is a well-known pest 
in Europe and North America where according to the phenology 
of its liliaceous host plants of the genus Allium, it enters the 
pupal diapause during August and September, and development 
is only resumed after winter, in May^. In Israel, in contrast, 
Allium plants under natural conditions germinate or sprout 
only after the first winter rains in November-December, and 
grow till the end of the rainy winter season, at the end of which 
they become domiant for the dry summer season. In close 
synchronization with this cycle the onion fly emerges in 
November and breeds one generation up to January. The 
majority of the offspring of the following generation enters the 
pupal diapause in March. This takes them through the dry 
period of absence of fresh Allium growth till November or 
December, depending on the first rain. A similar instance is 
that of the aphid-eating syrphid fly Epistrophe balteata de Geer, 
which in Switzerland, for example, diapauses as an adult during 
winter, when aphids are very rare^^, whereas the same species in 
Israel raises several generations during winter and spring, when 
aphids are abundant, and is dormant during summer when its 
prey becomes extremely scarce^^. 

One cannot assume that the factors which induce winter 
diapause in the onion or syrphid fly in the temperate zone are 
precisely the same ones that cause the same species to aestivate 
in a subtropical region, though the reason for arresting develop- 
ment is the same, namely, to survive a period of absence or 
shortage of food. This must lead one to suspect that the changes 
which take place within the host plant in anticipation of an 



DIAPAUSE AND INSECT PHENOLOGY 157 

impending crisis affecting its development (oncoming winter 
cold in the temperate zones, or the approach of dry summer in 
the semi-arid zones), must be communicated to the insects 
consuming those plants. In other words, either diapause- 
inducing or development-inhibiting substances are to be sought 
in the diet of insects, especially those exhibiting facultative 
diapause. This should be a mechanism somewhat similar to that 
of the response to the host in parasitic insects, although Dr. Lees 
will probably argue that the latter is a case of quiescence rather 
than of strict diapause. 

Before concluding I should like to raise briefly one more 
point. One might be highly tempted to try to dismiss the whole 
issue by the trite argument that we are dealing with quite 
distinct geographical or ecological races of the species; in the 
case of the codling moth different names have even been given, 
viz. Putaminana Stdgr. for the Near Eastern race. It might in 
turn be argued that each of these races hereditarily responds to 
a certain consistent environmental agency, whether as regards 
the onset of diapause or its termination, and not to alternative 
agencies, according to the different environments. Allow me to 
avert these temptations by quoting a few lines from an article 
written by Bodenheimer and Vermes^- some three years ago: 
'Diapause depends upon the interplay of heredity and environ- 
ment. Hence, no general genetical solution of the problem of 
diapause determination can possibly be given. Once the one, 
once the other factorial group may be the main inducing factor. 
Yet even under apparently similar environmental conditions 
during the critical period or in populations of an apparently 
higher homogeneous genetical constitution may result as 
different phenotypical manifestations.' 

REFERENCES 

^ A. D. Lees, The Physiology of Diapause in Arthropods, Cambridge Uni- 
versity Press, London, 1955. 
- E. RivNAY, Proc. 10th Intern. Congr. EntomoL, Montreal, 2 (1958) 743. 
^ L. BoNNEMAisoN, Ann. epiphyt., 11 (1945) 19. 



158 I. HARPAZ 

J Z. AviDOV, Ktavim, 2-3 (1952) 43. 

5 R. C. Dickson, Ann. Entomol. Soc. Am., 42 (1949) 511. 

^ I. Harpaz, Unpublished results. 

■ H. H. Clayton and F. L. Clayton, World weather records, 1931-1940, 

Smithsonian Institution, Washington, D.C., 1947. 
^ S. Yathom, Ph. D. Thesis, Hebrew University, Jerusalem, 1959. 
9 M. Miles, Bull. Entomol. Research, 49 (1958) 405. 

10 F. Schneider, Mitt. Schweiz. Entomol. Ges., 21 (1948) 249. 

11 I. Harpaz, Ph. D. Thesis, Hebrew University, Jerusalem, 1953. 

1- F. S. Bodenheimer and P. M. Vermes, Studies in Biology, Jerusalem, 
1 (1957) 106. 

DISCUSSION 

Lees: I do not think it profitable to argue whether it is an 
example of diapause or quiescence; everybody should be allowed 
to make up his own mind on this point. My question refers to 
Gambaro's work in Italy. She believes that the maturity and the 
variety of the apple has a considerable influence on diapause in 
the codling moth. Is your Baghdad race affected similarly? 

Harpaz: There is no synchronisation between the phenology 
of our apple trees and that of the codling moth. When the insect 
emerges before the fruit is available, it finishes its larval develop- 
ment entirely on leaves. 

Hestrin: You have suggested that the induction of diapause 
prior to nutritional deficiency is a useful characteristic; but how 
does selective pressure in evolution produce an anticipatory 
mechanism? 

Harpaz : This was only a suggestion. I feel it must be cumula- 
tive over a period of time, you cannot induce diapause by shock 
treatment. 

Lees: I do not see any great difficulty here. If natural selection 
weeds out the animals which do not enter diapause, 'antici- 
patory' mechanisms would surely be evolved rather rapidly. 



ECOLOGICAL PROBLEMS OF SEED DORMANCY 

D. ROLLER 

Department of Botany, 
Hebrew University, Jerusalem (Israel) 



Dormancy in seeds is a misleading term. A seed which does 
not germinate when placed in moist soil is just as dormant as a 
plant which does not flower during long days because it requires 
short days for flowering. The only dilTerence between the two is 
that in the case of the seed we are dealing with initiation of 
growth while in the other we are dealing with initiation of 
flowering. In both cases the process of initiation is triggered and 
controlled by environmental signals, and may be said to be 
regulated by the environment^. Some seeds require a period of 
so-called 'after ripening' under a specific range of environmental 
conditions before they are ready to germinate-^"^, and analo- 
gously many plants require a certain period of vegetative 
growth under a specific range of environmental conditions 
before they achieve 'readiness to flower'. Also, many seeds are 
known to become less dormant as they age, insofar as they 
germinate within a wider range of environmental conditions^' ^. 
and analogously many plants which require a certain photo- 
period or vernalization for flowering may eventually flower 
without them when they are sufficiently old. 

If we accept this analogy we may approach the question of the 
significance of these phenomena in the existence of the species. 
The common denominator for these phenomena is the existence 
of a biological system which permits a choice between starting 
a new developmental phase and continuing in the previous one. 
It is logical to assume that such systems have evolved as a result 
of their being of survival value to the species. Presumably a 
photoperiod-sensitive plant utilizes the most dependable time- 
telling device, the astronomical clock, to choose the time of 
flowering suitable for that particular species in its particular 
habitat to carry out to completion one essential part of its 

References p. 162 



160 D. ROLLER 

program of survival, namely formation of viable seed. The 
question under discussion here is if and to what extent regulation 
of germination performs a similar function in another part of the 
program of survival of the species, namely establishment of the 
seedlings in a suitable environment at a suitable time. 

Several germination regulating mechanisms have been identi- 
fied in seeds, for example control of water entry into seed, 
control of the gas exchange between the environment and the 
embryo, germination inhibitors, control by specific temperature 
response, control by light response. Some of these mechanisms 
may indeed be visualized as bearing ecological significance. 
Thus, the control of water entry into the seed, which is achieved 
by the presence of a water-impermeable layer surrounding the 
embryo-' ^' ^, may operate in two ways. The impermeability 
may disappear from a fraction of the seed crop at a time over a 
long period^' ^, or it may disappear simultaneously from almost 
the entire population through the action of some environmental 
factor (frost^o, heat^i, microbial activity in the soil^- or in the 
stomach of ruminants^^' i"^, or at specific relative humidities^^ 
or by action of fire^^). In the former case, the repeated 
attempts at establishment compensate for the haphazardness in 
choice of environment. In the latter case, specific environments 
would be favoured. 

Regulation by germination inhibitors has some clear ecological 
implications, in that certain environments are excluded and 
others are favoured^'^. Thus, inhibitors in pulp and juice of 
fleshy fruits exclude precocious germination inside the fruit^^; 
water-soluble inhibitors in dispersal units of desert plants may 
act as rain-gauges in preventing germination until a certain 
critical amount of rain has fallen^^"'-^; inhibitors may also aid in 
preventing over-population by seedlings (whether of the same or 
other species)-^. 

Control by specific temperature also has some obvious 
ecological implications. The simplest of these are the require- 
ments for specific ranges of more or less constant temperaf 
tures^s, 26_ ^ more sophisticated control is found in seeds o- 



ECOLOGY OF SEED DORMANCY 161 

temperate-zone plants, which require 'stratification', namely, 
pretreatment of the moist seed by cold^' ^. This restricts germi- 
nation not only to post-winter conditions, thus minimizing the 
dangers of frost killing, but also to regions with cold winters, 
which most of these plants require for other developmental 
phases (e.g. breaking of bud-dormancy, flowering). A similar 
argument may be postulated for seeds the germination of which 
is optimal at diurnally fluctuating temperatures^^* 27-29, 30-33 
It is likely that such a requirement confines germination to 
regions, seasons and soil depths where such fluctuations 
occur^' 20' ^^. 

Control by specific light requirements may operate in germi- 
nation in much the same way as it does in flowering, by providing 
the process with a celestial clock. Short day seeds and long day 
seeds have been described^-^' ^^. In addition, since the embryo is 
heterotrophic, light-inhibited seeds may also be considered short 
day seeds, while seeds which require even a flash of light are 
comparable to long day seeds. Seeds of many hygrophytes 
require light for germination^'^"^^. Here it is likely that the 
requirement places a limit to the water-depth at which germi- 
nation will occur 1. A similar mechanism may be operative in 
plants which form forest undergrowth. 

Several problems present themselves in studies of this nature. 
Two of these stand out because of their importance and the 
danger of ignoring them. One is the fact that the vast majority 
of our information on germination has been obtained by 
experiments on or between filter papers, in Petri dishes or 
germinators and using distilled water. Not only may some of the 
data be a result of these experimental artifacts, but data may 
have been overlooked. A case in point is that of lettuce seeds 
which at 20^ appear light-insensitive when germinated in water, 
but require light when germinated in solutions above a given 
osmotic value^o j\^q other important consideration is funda- 
mental to ecology. To what extent are the ecological implications 
more than rationalization of phenomena which have little, if 
anything, to do with the actual situation? It is dangerous as it is 

References p. 162 



162 D. KOLLER 

tempting to ascribe profound meanings to biological phenomena. 
Yet if such meanings actually exist their elucidation may be of the 
utmost importance. With regard to germination, we can only 
point to the general fact that cultivated species, in which 
dormancy has been selected against, require constant reseeding, 
while most wild species, in which regulated germination has been 
naturally selected for, are hard to eradicate by any single 
treatment. This fact is suggestive, but experimental techniques 
have to be devised for testing the hypothetical ecological 
implications of the so-called dormancy in seeds. 

REFERENCES 

1 D. KoLLER, Bull. Research Council Israel, 5 D (1955) 85. 

- L. V. Barton and W. Crocker, Twenty Years of Seed Research, Faber 

and Faber, London, 1948. 
^ W. Crocker, Growth of Plants, Reinhold, New York, 1948. 
^ W. Crocker and L. V. Barton, Physiology of Seeds, Chronica Botanica, 

Waltham, Mass., 1953. 
^ E. Brown, T. R, Stanton, G. A. Wiebe and J. H. Martin, U.S. Dept, 

Agr. Tech. Bull., (1948) 953. 
« V. Kearns and E. H. Toole, U.S. Dept. Agr. Tech. Bull., (1939) 638. 
' W. Crocker, Botan. Gaz., 42 (1906) 265. 
* G. T. Harrington, /. Agr. Research, 6(1916) 761. 
9 D. V. JuBY and J. H. Pheasant, /. EcoL, 21 (1933) 442. 

10 A. R. Midgeley, /. Am. Soc. Agron., 18 (1926) 1087. 

11 J. P. Jones, Mem. Agr. Exptl. St a. Cornell Univ., (1928) 120. 

12 N. E. Pfeiffer, Contribs. Boyce Thompson Inst., 6 (1934) 103. 

13 G. W. Burton, /. Agr. Research, 76 (1948) 95. 

1^ P. Mueller, Ber. schweiz. botan. Ges., 43 (1934) 241. 

1^ E. O. C. Hyde, Ann. Botany (London), 18 (1954) 241. 

1*5 E. C. Stone and G. Juhren, Am. J. Botany, 38 (1951) 368. 

17 M. Evenari, Botan. Rev., 15 (1949) 153. 

18 H. Oppenheimer, Sitzber. Akad. Wiss. Wien. Abt. I, 131 (1922) 279. 

19 D. KoLLER, Ecology, 38 (1957) 1. 

20 D. KoLLER AND M. Negbi, Ecology, 40 (1959) 20. 

21 D. KoLLER, N. H. Tadmor and D. Hillel, Ktavim, 9 (1958) 83. 

22 F. W. Went, Ecology, 30 (1949) 1. 

23 p \Y Went, Desert Research, Proc. Research Council Israel, Special 
Publ. No. 2, (1953) 230. 

2^ J. Bonner, Botan. Rev., 16 (1950) 51. 



ECOLOGY OF SEED DORMANCY 163 

-5 E. H, Toole and V. K. Toole, Proc. Intern. Seed Testing Assoc.., II 

(1939) 51. 
•26 F. W. Went and M. Westergraad, Ecology, 30 (1949) 26. 

27 W. A. Gardner, Botan. Gaz., 77 (1921) 249. 

28 G. T. Harrington, J. Agr. Research, 23 (1923) 295. 

29 D. KoLLER, Ph. D. Thesis, Hebrew University, Jerusalem, 1954. 

30 T. I. Morinaga, Am. J. Botany, 13 (1926) 141. 

31 E. H. Toole and V. K. Toole, /. Agr. Research, 63 (1941) 65. 

3- E. H. Toole, V. K. Toole, H. A. Borthwick and J. B. Hendricks, 
Plant Physiol., 30 {1955} 15. 

33 V. K. Toole, J. Agr. Research, 62 (1941) 691. 

34 F. W. Went, Ann. Rev. Plant Physiol., 4 (1953) 347. 

35 S. IsiKAWA, Botan. Mag. Tokyo, 67 (1954) 51. 

3 6 P. F. Wareing, Photoperiodism and Related Phenomena in Plants 
and Animals (R. B. Withrow, Ed.). Am. Assoc. Advancement Science, 
Washington, 1959, p. 73. 

3" L. V. Barton and J. E. Hotchkiss, Contribs. Boyce Thompson Inst., 12 
(1951)277. 

38 D. IsELY, Mem. Agr. Exptl. Sta. Cornell Univ., (1944) )257. 

39 N. H. Tadmor, D. Roller and E. Rawitz, Ktavim, 9 (1958) 177. 
'lo A. Kahn, Plant Physiol., 35 (1960) 1. 



DISCUSSION 

Lees : If the low temperature requirement for seeds is a kind 
of area restrictive device, I cannot see its value from the evo- 
lutionary point of view. Why should the evolution not be such 
that, while such a device is present in cold climates, the device 
gradually disappears when the plant moves to a warmer climate. 
This is what happens in insects. 

Koller: Certain varieties growing in moist regions require 
much higher rainfall to wash away inhibitors of germination 
than other varieties of the same plant growing in deserts. This 
may be a partial answer to your question in a parallel case. 

Mayer : What is the ecological or survival value of the Red- 
Far Red mechanism in germination and in flowering? 

Koller : The Red-Far Red system is just one part of the 
intricate 'biological clock' by which the organism recognizes the 
seasons. 



164 D. KOLLER 

Keynan: Is this light sensitivity not a means for the seed to 
know that it is at the right depth for germination? 

Koller: This is unlikely, as lettuce seeds planted in the soil 
are no longer light sensitive. 

Nachmony: The temperature range may be another expla- 
nation. Wareing found that the germination of Betula seeds at 
15° can be induced by photoperiodic treatment, while at 26° a 
flash of light was sufficient to initiate germination. 

Koller: In our laboratory germination was independent of 
light at 20° or below. 

Lees : Germination in the dark might be equivalent to germi- 
nation in continuous light. 

Galun : What is the ecological value of low oxygen tension? 

Koller: Marsh plants germinate only under marsh con- 
ditions. I would like to remark that we cannot generalize about 
'normal' conditions, since every variety has its conditions for 
germination, and the dormancy of the seed may be dependent 
on the availability of these specific conditions. 



ROLE OF PLANT HORMONES IN 
DORMANCY OF SEEDS 

ALEXANDRA POLJAKOFF-MAYBER 

Department of Botany, Hebrew University, 
Jerusalem (Israel) 

The importance ascribed to plant hormones in controlling 
various functions and processes of plant growth and behaviour 
is increasing rapidly. 

The first group of plant hormones isolated and identified was 
that of the auxins. These were discovered in connection with 
studies on stem elongation, and extension growth is still the 
basis of some bioassay methods for this group of hormones. 
They are sometimes designated as plant growth substances^' -. 
In this group are classified all the substances causing extension 
growth and having in their structure an unsaturated arom.atic 
nucleus and a side chain terminating in a carboxyl group or 
one that is easily oxidized to a carboxyl. The best known 
representative of this group is 3-indolyacetJc acid (lAA). 

As research was extended, it was proved that the same sub- 
stances actually aff'ect numerous and very variable aspects of 
plant development, such as root initiation, cambial activity, 
apical dominance, leaf abscission, fruit growth and many 
others^- 3. For many years the auxins were the only identified 
group of hormones; other hormones such as the 'calins' and the 
'florigens' were postulated, but not isolated or identified. 

Recently, two new groups of substances were isolated and 
their effect on plant development allows us to include them 
among the plant hormones; these are the gibberellins and the 
kinins. The gibberellins were first isolated as products of fungal 
metabolism^' 5. and kinetin, the best known compound among 
the kinins, was artificially produced from nucleic acids^. In due 
course it was shown that higher plants apparently contain 
substances of similar structure and activity. 

Evidence will be brought here to show that the three groups 

References p. 172 



166 A. POLJAKOFF-MAYBER 

of hormones have some role in regulating dormancy in seeds. 
There are also other substances found in the seeds but not as yet 
identified, which take part in regulating the dormancy phe- 
nomenon and germination processes. These substances are 
either germination inhibitors or germination promoters, and 
they are the products of normal metabolic processes in the 
ripening and in the germinating seed. Similar substances regulate 
the dormancy and sprouting of buds. 

As has already been explained in previous lectures, various 
dormant seeds will not germinate unless their dormancy is 
broken by special temperature or light treatment. In some cases 
such treatment may be avoided and the seeds may be induced to 
germinate by treating them with plant hormones. Such seeds, 
for example, are lettuce seeds [Lactuca sativa L.) variety Grand 
Rapids. These seeds, for several years after harvesting, will not 
germinate in the range of temperatures of 23-28 "" unless 
illuminated, when fully imbibed, for a certain period of time 
with red or white light. This light requirement diminishes with 
increasing period of storage, and five to six years after harvesting 
the seeds will germinate in the dark to the extent of 80% or 
more. Irradiation with Far Red reverses the eff'ect of red and 
usually also lowers the percentage of germination in the dark. 

The red light treatment breaks the dormancy of the seeds and 
forces them to germinate. A similar effect may be achieved by 
soaking the seeds in a solution of gibberellin''' ^' ^. The mecha- 
nisms of the two treatments are not identical as the effect of 
both of them is additive (Table I) and the Far Red completely 
reverses the red effect but only partially the gibberellin effect^. 

Besides in lettuce, gibberellin has been shown to be effective 
in breaking the dormancy of Arabidopsis'^^, Kakmchos^^ and 
many other species^-- ^^. 

A closer interaction between the effects of light and a plant 
hormone in breaking dormancy was shown for kinetin^^' ^^. 
Kinetin greatly increases the sensitivity of the seeds to red light. 
In the presence of kinetin, extremely small amounts of light 720 
ft.c.s. (foot-candle-seconds) are sufficient to bring about maximal 



PLANT HORMONES AND SEED DORMANCY 167 

TABLE I 

THE EFFECT OF LIGHT AND GIBBERELLIN TREATMENTS ON THE GERMINATION OF 

LETTUCE SEEDS 

Results given as percent germination. The red illumination was given after 
two hours' imbibition. The Far Red was given either immediately after the 
Red or, when given alone, after 30 min of imbibition. The concentration 
of the gibberellic acid was 2.9 x lO^' M '. 







Gibberellic 




Water 


acid 


Dark 


12 


39 


Red 


44 


66 


Far Red 


5 


25 


Red and Far Red 


11 


35 



germination, while in the absence of kinetin 3600 ft.c.s. are 
necessary to bring about the same result^^. 

In all these cases it is not clear what are the actual mechanisms 
that are affected either by light treatment or by the hormones. 

The possibility that lAA may play a part in the germination of 
seeds has been considered for many years, but the results of the 
experimental work were rather contradictory. Some suggested 
an inhibition of germination by excess of auxins present in the 
seedsorinthefruits^^' i^, while others suggested stimulation^^' ^^. 
Soding and Wagner^^ tried to settle this controversy by suggest- 
ing that lAA stimulates the germination of dormant seeds but 
does not affect the germination of non-dormant ones. They 
tried unsuccessfully to test this hypothesis on Poa seeds but 
the idea proved to be correct for lettuce seeds^^"-^' ^^. The 
more dormant the seeds were, and the lower the temperature 
during germination, the more effective was lAA in breaking the 
dormancy and in stimulating germination. 

The next step was, therefore, to find out whether the seeds 
contain any endogenous lAA and whether its amount changes 
during germination. Dry seeds and seedlings of lettuce of varying 

References p. 172 



168 



A. POLJAKOFF-MAYBER 



ages were extracted and the extracts fractionated and separated 
with the aid of paper chromatography using the conventional 
methods for lAA isolation'-. The various parts of the chromato- 
gram were assayed using the extension growth bioassay. The 
results are summarized in Fig. 1. 



140 
120H 



100 



140- 
120 



D 100 



100 



J L 



140- 
120t^^^ 



._^j^r^ 




01 



12 



24 



t_ 

D 

o 



48 



72 



96 



100 



O 0.1 0.2 03 0.4 0.5 0.6 0.7 0.8 0.9 1.0 /?.. 



Fig. 1. Histograms showing the chromatography separation of acid 

growth-active substances in lettuce seeds germinated for various lengths 

of time. The biossay used was the oat coleoptile extension growth test. 

The broken Hne gives the growth of the controls—. 



It is evident that dry seeds do not contain any lAA or any 
other acid growth promoter. They do contain growth inJiibitors. 
lAA appears between Rf 0.35-0.45, when the seedling already 
shows appreciable development. 

Parallel to our work on the relation between growth promoting 
and growth inhibiting substances and dormancy, other groups 
have been engaged on the same problem. 



PLANT HORMONES AND SEED DORMANCY 169 

Varga and her associates''^ and also Hemberg23-25^ ^^e mainly 
interested in the mechanism regulating the dormancy of potato 
tubers, while Wareing and his group^^ are interested mainly in 
the dormancy of seeds and buds of woody plants. These are the 
principal groups working on the subject but there are many 
other investigators working in this field. 

In all these investigations it very soon became clear that it is 
not possible to draw any definite conclusion, regarding the 
germination of seeds or the sprouting of buds, while using the 
extension growth bioassay. Special bioassay tests were, therefore, 
devised for every case. 

Hemberg-3"-^ ascribed the dormancy of the potato tubers to a 
growth inhibitor present in the potato peel. Blommaert'^^ and 
Varga and Ferenczy^^ isolated this inhibitor and showed that 
its amount decreases as the tubers emerge from dormancy. But 
when this inhibitor was tested in a potato sprouting test'-^, it 
was shown to have no effect. 

More convincing results were achieved by Wareing and his 
group-^. They showed that the dormancy of Xanthium seeds is to 
a large extent due to the presence of an inhibitor which prevents 
the growth of the embryo. Germination proceeds only after 
this inhibitor is either leached out or oxidized inside the seed. 
They have also shown that the dormancy of Fraxinus seeds^^ is 
regulated by an interplay between growth inhibitors and germi- 
nation stimulators. The inhibitors present in the endosperm and 
the embryo itself, are probably the main cause of the dormancy 
phenomenon. During stratification there is no change in the 
inhibitor's content, but a germination stimulator is formed which 
counteracts the effect of the growth inhibitor and brings about 
the breaking of dormancy, thus enabling germination (Fig. 2). 

Similar investigations were carried out in our laboratory with 
lettuce seeds^^. The same light-sensitive variety (Grand Rapids) 
was used. The seeds were imbibed in water, in the germination 
inhibitor — coumarin, or in the germination stimulator — 
thiourea. Germination inhibitors and germination stimulators 
were followed up in all the three series (Fig. 3). It is evident that 

References p. 172 



170 



A. POLJAKOFF-MAYBER 



IOt 
8 

6' 

4- 







_, , ^ 



-HgO 



0.2 0.4 0.6 as 1.0 



IOt 
8 

6f 

4 
2 
O.. 



B 



■HgO 



0.2 0.4 0.6 0.8 1.0 



Fig. 2. Histograms showing the chromatographic separation of unchilled 

and chilled Fraxinus seeds. The bioassay used was Fraxinus embryo 

germination test. The broken line gives the water controls ^°. 



dry lettuce seeds and seeds imbibed in water in the dark contain 
germination inhibitors. On imbibition in water, the acid 
inhibitors disappear just before the actual germination begins, 
the neutral inhibitors being still present. This may explain the 
low germination percentage in the dark. On imbibition in 
coumarin, coumarin accumulates in the seeds^^ and apparently 
blocks germination. Comparison of the various histograms 
suggests that some of the natural inhibitors may also be coumarin 
derivatives. In another case^^ a natural germination inhibitor 
present in the seeds of TrigoneUa arabica was identified as 
coumarin. 



60 
40 
20 



60- 
40- 
20 



60 

401 

20 



60 
40 
20 

60- 
40- 
20- 

60- 
40- 
20- 



A 



-10 





.-8 




7 



.--6 




't'^Wi 



--^//-. 



B 



^^^""^m^ 



-10 



la. 




8 






5 ._-.J-,,^.__.5 




^-^ 



"J~ 



-^^C^- 



-2 



^"^^^"'"^ ""ll^^'^^^"""' 



—I 1 r- 



0.2 04 0.6 0.81.0 



r 



0.2 04 0.6 0.8 1.0 



Fig. 3. Histograms showing the chromatographic separation of substances 
affecting germination extracted from lettuce seeds germinated in water, in 

coumarin or in thiourea. 
A — acidic extracts; B — neutral extracts; 1 — dry seeds; 2 — seeds 
imbibed for 2 h in water in the dark; 3 — seeds imbibed for 4 h in water 
in the dark ; 4 — seeds imbibed for 12 h in water in the dark ; 5 — coumarin 
control; 6 — seeds imbibed for 2 h in coumarin; 7 — seeds imbibed for 
12 h in coumarin; 8 — thiourea control; 9 — seeds imbibed for 2 h in 
thiourea; 10 — seeds imbibed for 12 h in thiourea. The broken lines give 

the water controls ^^. 



172 A. POLJAKOFF-MAYBER 

Thiourea, a germination stimulator, apparently changes the 
ratio between the various natural substances affecting germi- 
nation, as it induces the formation of germination stimulators. 
This apparently occurs during the initial phases of imbibition. 
This change may initiate a chain of reactions eventually leading 
to germination. 

To sum up the facts presented here, it seems that the plant 
hormones do affect germination, perhaps by changing the initial 
ratio between the natural germination or gi-owth stimulators and 
inhibitors. This idea is supported by the facts that gibberellin 
counteracts the inhibitory effect of coumarin^^ ^j^^j ^j^^^ lettuce 
seeds do contain appreciable amounts of gibberellin-like 
substances^^. The natural inhibitors and stimulators are as yet 
not identified and we do not know whether they may be classified 
as hormones. A more complete study is required for the identi- 
fication of these substances and for the evaluation of their 
changes under various treatments affecting germination. Al- 
though it seems possible that the factors involved in dormancy 
exercise their effect through the internal equilibrium of such 
hormones, it is as yet by no means understood which are the 
basic reactions affected by the hormones and it is as yet not 
certain that this is the only mechanism involved. 

ACKNOWLEDGEMENTS 

Our thanks are due to Prof. P. F. Wareing and to Nature for 
their permission for the use of Fig. 2 and to Dr. S. Blumenthal- 
Goldschmidt for her permission for the use of Fig. 3. 

REFERENCES 

1 L. J. AuDUS, Plant Growth Substances, Leonard Hill, London, 1959. 

2 T. Weevers, Fifty Years of Plant P/i>'5/o/o^v, Chronica Botanica U.S.A., 
1949. 

^ A. C. Leopold, Auxins and Plant Growth, University of California Press, 
1955. 

4 P. W. Brian, S.E.B. Symposium, XI, (1) 1957, 166. 

5 B. B. Stowe and T. Yamaki, Ann. Rev. Plant Physiol, 8 (1957) 181. 



PLANT HORMONES AND SEED DORMANCY 173 

'^ F. Skoog and C. O. Miller, S.E.B. Symposium, XI (1957) 118. 
■ M. EvENARi, G. Neumann, S. Blumenthal-Goldschmidt, A. M. 
Mayer and A. Poljakoff-Mayber, Bull. Research Council Israel, 6 D 

(1958) 65. 

8 A. Kahn, J. A. Goss AND D. E. Smith, Science, 125 (1957) 645. 

'' F. LoNA, Ateneo parmense, 27 (1956) 641. 
1" F. J. Kribben, Naturwiss., 44 (1957) 313. 
11 R. Bunsow and K. von Bredov, Naturwiss., 45 (1958) 46, 
1- P. KoLLio AND p. PiiROiNEN, Nature, 183 (1959) 1830. 
^^ R. Leizorowitz, M. Sc. Thesis, Hebrew University, Jerusalem, 1960. 
1^ C. O. Miller, Plant Physiol., 33 (1958) 1 15. 

15 J. Weiss, personal communication. 

16 C. Izard, Compt. rend., 242 (1956) 2027. 

1" S. Naik, /. Indian Botan. Soc, 33 (1954) 153. 

18 A. Gerrard, New Phytologist, 53 (1954) 105. 

19 HoFFSCHLAG, as citcd by H. Soding^^. 

20 A. Poljakoff-Mayber, Bull. Research Council Israel, 6D (1958) 78. 

21 A. Poljakoff-Mayber, A. M. Mayer and S. Zacks, Ann. Botany 
(London), 22(1958) 175. 

22 A. Poljakoff-Mayber, S. Blumenthal-Goldschmidt and M. Evenari, 
Physiol. Plantarum, 10 (1957) 14. 

23 T. Hemberg, Arkiv. Botany, 33 5 (1946) 1. 

2 1 T. Hemberg, Physiol. Plantarum, 2 (1949) 24. 

25 T. Hemberg, Physiol. Plantarum, U (1958) 615. 

26 K. L. J. Blommaert, Nature, 174 (1954) 70. 

2" M. B. Varga and L. Ferenczy, Acta Botanica Acad. Sci. Hung., 3 
(1957) 11. 

28 M. L. Buch and O. Smith, Physiol. Plantarum, 12 (1959) 706. 

29 P. F. Wareing and H. a. Foda, Physiol. Plantariun, 10 (1957) 266. 

30 T. A. ViLLiERS and p. F. Wareing, Nature, 185 (1960) 112. 

31 S. Blumenthal-Goldschmidt,/*/?. D. Thesis, Hebrew University, Jerusa- 
lem, 1959. 

32 A. M. Mayer, Physiol. Plantarum, 6 (1953) 413. 

33 H. R. Lerner, a. M. Mayer and M. Evenari, Physiol. Plantarum, 10 

(1959) 245. 

3"! S. Blumenthal-Goldschmidt and A. Lang, Nature, 186 (1960) 815. 

35 A. M. Mayer, Nature, 181 (1959) 826. 

36 H. SoDiNG and M. Wagner, Planta, 45 (1955) 557. 



174 A. POLJAKOFF-MAYBER 

DISCUSSION 

Keynan : You mentioned, that by using a different part of the 
Hght spectrum you can 'unswitch' tiie stimulation of germination 
by red light. However, after about two hours this 'unswitching' 
is no longer possible and germination proceeds. Is there any 
visible or measurable change during these two hours? 

Poljakoff-Mayber : Our methods are not sufficiently refined 
to allow an answer to your question. 

GoLDWASSER : What is the range of concentrations at which 
these inhibitors and stimulators act, and what quantities do you 
succeed in isolating? 

Poljakoff-Mayber: While we can extract quantities sufficient 
to obtain the necessary effects, these quantities are not sufficient 
for measuring purposes. 

Goldwasser: Well, in that case you ought to be able to 
measure the changes occurring in the seeds indicated by Dr. 
Keynan. 

Poljakoff-Mayber: We hope to succeed in doing so, once 
we have refined our methods sufficiently. 



DORMANCY AND 
DORMANCY BREAKING IN SEEDS 

S. KLEIN 

Department of Botany, Hebrew University, 
Jerusalem (Israel) 

It would be very helpful if one could start a lecture like this 
with a clear and physiologically valid definition of what is meant 
by dormancy. The fact that it was thought desirable to compare 
this phenomenon in various groups of organism shows that we 
are still far from a general formula. In the broadest sense, 
dormancy in seeds means the cessation of growth in the embryo 
without the latter losing its viability for a prolonged time. The 
embryo may or may not be enclosed in its natural envelopes. 
These may consist of an endosperm, a seedcoat and other 
surrounding layers, which actually do not belong to the 'seed' 
proper. Since they may all influence the behaviour of the embryo 
it is more appropriate to talk about dormancy in 'dispersal 
units' than in seeds^. 

It is evident that such a period of dormancy is of extreme 
importance in the life of the plant. It allows effective dispersal 
of the young plants both in space and time, and allows the 
embryos, which are extremely sensitive in the active state, to 
overcome conditions unfavourable to development and growth. 

It is also evident that dormancy is closely related to the 
subsequent renewal of growth. The processes which make this 
transition possible are therefore of importance in the under- 
standing of dormancy, and we shall be concerned principally 
with changes that occur during dormancy breaking and with 
differences before and after, i.e. between seeds before and during 
germination. 

This introduces another difficulty, namely, to define what is 
meant by germination. In order to renew its normal activities, 
the dormant seed has to be exposed to certain external conditions, 
e.g., a certain temperature range, water, a certain oxygen level. 

References p. 190 



176 S. KLEIN 

Frequently the presence of these factors is not sufficient, and 
other conditions have to be fulfilled. Given the necessary 
environmental conditions, growth will be resumed after a 
certain time, and the radicle, or in some cases the hypocotyl, 
will start to penetrate through the coverings of the dispersal 
unit; from then on processes of growth and development will 
usually continue. 

We define as germination processes those which occur from 
the moment the factors necessary to break dormancy are 
supplied until the time when growth is resumed (considerable 
time may elapse before the radicle protrudes through the seed- 
coat-""*). This allows us to differentiate between processes 
occurring during resumption of growth and continuation of 
growth, a distinction the usefulness of which has been pointed 
out repeatedly^. Unfortunately, since the most spectacular point 
in all of these processes is the protrusion of the radicle, there has 
been a tendency to regard this as the starting point of germination. 
Many papers dealing with 'changes occurring during dormancy- 
breaking' actually describe changes occurring during growth. 

The study of seeds which germinate only after some special 
condition is fulfilled throws light on particular steps of the 
germination process^. Let us, therefore, examine some of the 
different factors which may be necessary for the germination of 
seeds (for a more complete coverage of this topic, see^' ^' ^). 

Removal of, or treatment of the surrounding layers will in 
some cases be effective. The reason for the incapacity of the 
embryo to develop, even when the three 'primary' requirements 
— water, oxygen, optimal temperature — are present, may be 
located not in the embryo itself, but in the sun'ounding coats, 
and by removing them completely or partially, or by otherwise 
treating them, the germination block may be overcome. The 
ways in which the surrounding layers may influence the embryo 
are various : the layers may be hard and impermeable to water, 
as in the seeds of the Leguminosae, thereby pi eventing germi- 
nation. Or they may decrease gaseous exchanges as e.g. in the 
cocklebur and many grasses. 



SEED DORMANCY AND DORMANCY BREAKING 177 

In another group, the coats of the dispersal unit exert their 
influence through substances which inhibit the growth and 
development of the embryo^' ^. These inhibitory substances 
have actually been found everywhere in the dispersal unit, 
including the embryo itself, but on this latter point we shall hear 
in more detail from Dr. PoljakolT. However, in many cases, the 
embryo germinates readily after excision though it fails to 
germinate while enclosed in its coats because of inhibitory 
substances present in the endosperm or other layers. In such 
a case, even a small piece of endosperm adherent to the embryo 
may prevent it from growing. The natural substances cited as 
inhibitory include alkaloids, unsaturated lactones, unsaturated 
acids, ammonia, KCN and many others. It is, however, not 
always clear whether the inhibitory substance has a specific 
activity or whether the resulting inhibition is an osmotic effect. 
Lerner, Mayer and Evenari^ have shown that both effects may 
occur simultaneously, the main effect being either osmotic or 
specific. 

In a large variety of seeds, the inability to germinate cannot 
be ascribed directly to the influence of the seed coats, and even 
if the coats may be involved in the chain of events which 
eventually lead to germination, a treatment has to be given 
which will affect the embryo itself. This treatment may be 
prolonged dry storage, at the end of which a seed which did not 
germinate when imbibed immediately after harvesting will now 
do so. In order to break the dormancy during this process of 
after-ripening, different temperatures may be needed at different 
times^. 

In still other cases, a period of moisture at low temperature 
may be needed to awaken the embryo, a process which is known 
as stratification. With other seeds, alternating temperatures may 
be needed. The similarity between these temperature require- 
ments and those for breaking the diapause in insects are 
obvious^. 

In many cases, seeds have a strict light requirement which is 
influenced by temperature (for review on this aspect, see^- ^' i^). 

References p. 190 



178 S. KLEIN 

Within certain temperature ranges the seeds have a high germi 
nation percentage only in darkness or after exposure to a certain 
amount of light, both qualitatively and quantitatively definable. 
In a number of seeds, furthermore, a photoperiodic regime can 
increase germination. 

What makes matters complicated is that frequently, in a given 
dispersal unit, not one mechanism only is working but many 
are coupled together, so that very restricted conditions have to 
pertain to allow the seeds to germinate. To gain an under- 
standing of this aspect the problem is best approached ecolo- 
gically and we shall hear more about this from Dr. Koller. 

Another complication is that although the embryo as a whole 
is a biological unit, various parts of it may respond differently. 
A certain treatment may allow growth of one organ, let us say 
the radicle, but may not be sufficient to allow further devel- 
opment of the hypocotyl. Again, this requires the existence of 
different and specified conditions at various times. 

All this shows that the state of dormancy is a question of 
equilibrium between various mechanisms and not a case of 
one mechanism alone being blocked, or of the presence or 
absence of a certain substance. One should study, therefore, the 
interaction of various phenomena previous to, and after, 
dormancy-breakage, in order to get an overall picture of what is 
going on in a certain organism; the drawback being that a 
particular organism may be well fitted to give information on 
one aspect but not on another. 

In order to examine details of a single factor, for example, 
light-mechanism, it has been worthwhile to study this mechanism 
in different organisms, and not only in regard to germination 
alone. On the other hand, when nitrogen-metabolism is studied 
in seeds rich in protein, and carbohydrate-metabolism in those 
rich in starch, there is a certain danger in transferring con- 
clusions from one type of seeds to the other. 

We have concentrated our efforts on a single object, the 
lettuce seed, where all the aspects are equally difficult to study. 
Instead of giving examples of differences between states 



SEED DORMANCY AND DORMANCY BREAKING 1 79 

before and after germination from various plants, let me tell 
you something of the changes known to us to occur in one single 
organism, the light-sensitive lettuce seed. 

The dispersal unit of the lettuce seed, an achene, is composed 
of an embryo, the cotelydons of which serve as storage organs, a 
two-layered endosperm, a seedcoat, and a thick-walled fruit 
coat which, in case of the light-sensitive variety which will be 
discussed here, contains a brownish-black pigmental. 

When the seed has been imbibed in water, germination 
depends both on light and temiperature conditions. Below 
approximately 18% seeds germinate to a high degree in darkness, 
and light, therefore, increases the germination percentage only 
to a small extent. 

Between approximately 20-28'' the seeds require light for a 
high percentage of germination and remain dormant in the dark. 
The light requirement is strongest in freshly harvested seeds, and 
decreases with age of the seed. A mechanical treatment, such as 
removing or pricking the coats and the endosperm, or chemical 
treatment with e.g., thiourea, will cause germination even in 
the dark. 

Above approximately 30°, dormancy cannot easily be broken 
by light, but germination will result after various other treat- 
ments such as stratification for a few days previous to imbibition, 
high oxygen pressure or, again, removal of the endosperm. 

One of the most effective ways of overcoming the thermo- 
dormancy of lettuce seeds was found by Thornton-^ who states 
that lettuce seeds at 35° will germinate normally in an atmos- 
phere of 40-80 %C02. 

We can already see here the interaction of various mecha- 
nisms^o. At low temperatures, a photomechanism is taking place 
indicated by the slightly higher germination of illuminated seeds. 
At the same time other factors are at work which allow the seed 
to germinate in darkness. At higher temperatures, these factors 
cannot any longer break the dormancy, which however can still 
be overcome by the light mechanism. At still higher temperatures 
even this mechanism, is inhibited. Furthermore, the fact that 

References p. 190 



180 S. KLEIN 

dormancy can be broken by changes in gas pressure or by 
removal of the endosperm points to this structure as a factor in 
dormancy. I shall return to the influence of the seed coats on 
germination of the lettuce seed later. 

Part, if not all, of the light mechanism is connected with the 
well known reversible Red-Far Red reaction. The light effective 
in bringing about germination is red light of about 6500-6800 A, 
the promoting effect of which can be completely overcome by 
illumination by Far Red light, of wave length 7200-7500 A, and 
vice versa^' '^' i^. It is the same reaction that is active not only 
in the control of germination of a large number of seeds, but 
also in a wide array of other phenomena and can be regarded 
'as a general factor of growth control'", thereby relating seed 
germination to other biological phenomena. 

In this reaction, a single relatively short illumination is 
necessary to bring about the promoting or inhibitory effect. The 
sensitivity to this illumination depends on a number of factors, 
among others the time lapse between imbibition in darkness and 
illumination. At a temperature of 26°, lettuce seeds start to 
respond to light as little as approximately 10 min after imbibi- 
tion and the sensitivity rises for approximately 8 h, and then 
decreases^^. This time curve is not typical for all light-sensitive 
seeds. For Amarauthus seeds it was shown that several days are 
needed before sensitivity reaches its peak, but in this case the 
sensitivity is maintained even after two months^"^. 

As regards illumination with white light, a distinction should 
probably be made between the effects of a single short illumi- 
nation and those of prolonged continuous illuminationi^. For 
example, Kadman-Zahavi^-^ found in Amarauthus retroflexus, at 
a certain light intensity, 32% germination in the dark, 3% in 
continuous light and 92% after a single short illumination. In 
general, the main difference seems to be that in the case of short 
illuminations with white light, germination rises with light 
intensity, whereas with continuous illumination, germination 
response is inversely proportional to it^^. i5. Attempts have been 
made to explain this, assuming that the Far Red radiation 



SEED DORMANCY AND DORMANCY BREAKING 181 

present in 'white' light may be responsible for this, since it has 
been found that short and prolonged Far Red irradiations differ 
in their effect. In the Amaranthus seeds, a short illumination 
with Far Red is reversible by a subsquent red irradiation, 
whereas the inhibition by Far Red given over a prolonged period 
is not reversed by an immediately following illumination with 
red light^^. Therefore, when white light is given over a prolonged 
period, the 'prolonged Far Red' effect may be dominant over the 
stimulatory red effect and cause the inhibition. 

The effect of the prolonged Far Red irradiation, however, 
decreases with time : as mentioned, red light given immediately 
after the prolonged Far Red will be without effect: but when the 
seeds are kept for some time in darkness after the Far Red 
illumination and only then exposed to red-light, germination 
will occur. 

A somewhat similar difference between short and prolonged 
Far Red irradiation has been found also in certain lettuce seeds. 
The seeds of the 'Progress' variety are light sensitive only 
when very young. Older seeds have no light requirement and 
when imbibed, will germinate readily both in the dark and in the 
light. Red light has no effect on those seeds, because of the 
already high 'dark germination', but also a short Far Red 
illumination is without effect and will not cause inhibition of 
germination. However, when the seeds are exposed to prolonged 
Far Red irradiation, their germination is almost completely 
inhibited. This inhibition is reversible by red in-adiation but 
much stronger doses of red light are required than in the case of 
the usual 'Red-Far Red reaction' (Table I). Both the facts, that 
in this case (1) a short Far Red irradiation is without effect, 
whereas prolonged Far Red irradiation inhibits, and (2) that 
Red in much stronger doses than usual is needed to overcome 
the Far Red effect, may be taken as an additional indication 
that the 'prolonged Far Red' effect is different from the effect 
of Far Red in the well known short Red-Far Red reactionio. 
Still, it has yet to be shown definitely whether this difference 
exists in reality or whether the mechanism of the short Red-Far 

References p. 190 



182 S. KLEIN 

TABLE I 

GERMINATION PERCENTAGES OF LETTUCE SEED 'PROGRESS' 

illuminated with Far Red for 2(FR2), SCFRa) and 7 days (FR-) and then 

transferred to darkness (D) or illuminated with red (250 ft.-c.) for 5(R5), 

20(R2o) or 40 seconds (R40) (Evenariio). 





FR2 


FR5 


FRi 


D 


3 


4 


6 


R5 


10 


9 


6 


R20 


81 


27 


19 


R40 


80 


31 


26 



The germination of the untreated seeds in darkness varies between 90-93% 
(ref.io). 

Red reaction may not, after all, be responsible also for the 
above mentioned phenomena. The case of the Progress seeds is 
interesting also from another point of view. When these seeds 
are imbibed in a solution of coumarin, they behave exactly like 
the light-sensitive Grand Rapids variety!^. Thus, in cases in 
which one does not normally find light sensitivity, under certain 
conditions light may still have a profound influence. 

What makes understanding of the light mechanism still more 
difficult is that blue light too, in many cases, has a distinct 
influence, the nature of which is yet unclear. 

Without any doubt, however, the most important light 
mechanism is the reversible 'short' Red-Far Red reaction. Until 
very recently, attempts at identification of the pigment respon- 
sible for this reaction were unsuccessful. A few months ago, the 
photometrical detection of this pigment in living tissue of 
maize shoots was reported by Butler, Morris, Siegelman and 
Hendricks^''. The same group also succeeded in separating the 
pigment from the tissue by methods of protein chemistry. A 
spectral shift could be observed after both Red and Far Red 
irradiation. This is a most important advance since it enables us 



SEED DORMANCY AND DORMANCY BREAKING 183 

to tackle the nature of the enzymatic action involving this 
pigment through which germination and growth process in 
general are controlled. 

Among the many data available on light mechanism in the 
lettuce seed, I shall mention only two: First, that light exerts an 
influence not only on the imbibed, but also on the 'dry' seed^^. 
This influence, which is strongly dependent on the relative 
humidity of the air, is probably related to the internal water 
content of the seeds. Seeds stored at a relative humidity of 
60-80% in the light and subsequently germinated in the dark 
gave a higher germination percentage than those stored at the 
same relative humidity in the dark. No such effect of light could 
be found in seeds stored at 20-30% relative humidity. This 
indicates that the relatively low water content of the dormant 
seed has to be raised only slightly in order to make the seed 
respond to external influences. 

Secondly, the reversibility of the Red-Far Red reaction in the 
lettuce seed depends on the time interval between the two 
illuminations. After 10 min of darkness, Far Red cannot any 
longer reverse the effect of red^^. This shows that only the first 
steps in dormancy breaking are influenced by light. Once the 
reaction chain has started, light, at least in lettuce, does not have 
any influence on further events. 

Considering the changes induced by these light mechanisms 
which finally cause resumption of growth, one of the most 
important factors is, of course, water uptake. It might be 
imagined that dormant and non-dormant seeds would take up 
different amounts of water when imbibed, i.e. their water uptake 
would differ in lisht and darkness. This, however, is not the 
case, and water uptake under both these conditions proceeds 
almost indistinguishably until growth starts in the light-treated 
seeds, after approximately 14 h; i.e., the light treatment does not 
change water uptake in the seeds. 

Since a good deal of work has been done on chemical inhibi- 
tory and promotory agents for germination, let me say a few 
words on the effect of some of these substances in lettuce seed 

References p. 190 



184 



S. KLEIN 



100 

90 
c 
O 60- 

O 70 



£ 60 



1 A 



-o^ 




5 7.5 10 12.5 15 
mg % DNP 




12.5 15 

mg 7o DNP 

Fig. 1. Germination percentage of lettuce seeds (Grand Rapids) in different 

concentrations of dinitrophenol. 1 A: absolute germination percentages; 

1 B: germination percentages related to the germination percentage in water 

as 100. ( ) after illumination; ( ) in darkness. 



100 

c 90 
O 

■.^; 80 
o 
.E 70 

E 60 
*^ 50 
o 40 




1.0 2.0 3.0 4.0 
mg "/o Coumarin 




2.0 2.5 3.0 3.5 4.0 4.5 5.0 

mg "/o Coumarin 



Fig. 2. Germination of lettuce seeds in different concentrations of coumarin. 

Explanation as in Fig. 1. 



germination. Comparing the effect of coumarin with that of 
dinitrophenol, both of which are germination inhibitors, Figs. 
1 and 2 show the amount of seeds germinating as a function of 
various concentrations after a Hght treatment and in darkness. 
Since the percentage of germination of light-treated seeds is 
always higher than of seeds kept in darkness, this seems to 
indicate that light can to some extent overcome the effects of 



SEED DORMANCY AND DORMANCY BREAKING 185 

both the inhibitors. However, if we take the germination of the 
controls (in water) as 100, and express germination in the 
presence of the inhibitors as percentages of the water control, 
we see that the inhibitors differ in their action. Higher concen- 
trations of dinitrophenol depress germination equally in dark- 
ness and after light treatment, while coumarin at all concen- 
trations inhibits germination much more strongly in the dark, 
showing that light can to a certain extent obliterate the effect of 
coumarin. Since, as was mentioned earlier, the light mechanism 
influences only the early steps in the chain of reactions leading 
to germination, it would follow that coumarin acts on these 
early steps, whereas dinitrophenol affects processes which are 
not any longer under the control of the light mechanism. 2-4 D, 
according to Evenari, behaves in a similar way to coumarin^^. 

Since the question of respiration occupies so eminent a place 
in the symposium, let us say something about respiration in dry, 
resting seeds and in germinating ones. Do resting seeds respire 
or not? There seems uo be no doubt that the level of respiration 
in 'dry' seeds is largely a matter of moisture content. In general, 
down to a water content of approximately 10%, there always 
seems to be a small but measurable CO2 output and O2 uptake. 
When moisture is reduced below this level, the gas exchange is 
dramatically slowed down, and very frequently cannot be 
measured, although the seeds remain viable for a long time. As 
has been pointed out frequently, this only means that no 
measurable amount of CO2 can be detected and not that gas 
exchange actually ceases. 

James^^ cites in this regard a comment of F. F. Blackman: 
'It may well be that there will not be enough CO2 produced to 
be detectable in ten years, but who shall say that change has 
ceased? Our methods of analysis which demand a large aggregate 
of molecules for any demonstration are incapable of settling 
this philosophical question'. Even in those cases where small 
traces of CO2 output can be measured, two important questions 
arise. First, if this CO2 output is indicative of what usually is 
defined as respiration and secondly, how far it may not be due 

References p. 190 



186 S. KLEIN 

to micro-organisms. James cites Blackman as having considered 
that the CO2 output may be due to a purely photochemical 
reaction, in which the organisation necessary to maintain 
viability is destroyed, since viability is lost without any consider- 
able depletion of seed reserves. James concludes: 'So far as 
dormant seeds are concerned we might therefore still be up the 
horns of the old dilemmia: either they live without respiration, 
with extreme sluggishness no doubt, but still live, or, as Claude 
Bernard thought, they cease to live and come alive again'. 

However, when we consider the respiration of wet seeds, we 
are on firmer ground, since water uptake is always accompanied 
by an increase in respiration. During the first hour of imbibition 
there is a sharp rise in respiration, frequently with large and 
abrupt changes of RQ, giving quite extreme values. It has 
been shown, however, that these changes are due more to the 
physical conditions under which the seeds are imbibed than to 
changes in the respiratory apparatus. Later on, a more or less 
steady trend is reached, until the time when growth processes 
are resumed. In cases where no renewal of growth occurs, 
respiration rate goes down with a decrease of viability in the 
seeds^^. 

Is there any difference between the respiration rate of imbibed 
dormant and non-dormant seeds? Let us again consider lettuce 
seeds for illumination of this problem. Both dormant and non- 
dormant seeds can be compared under exactly the same con- 
ditions. The seeds are imbibed in darkness in Warburg flasks, a 
dose of light is given after two hours to certain of the flasks and 
readings are made. Since the first single mitotic division occurs 
in the germinating seeds after approximately 12-14 h, only a 
short time before the radicle protrudes through the seed coat, 
there is ample time to look for differences between dormant and 
non-dormant seeds during time of germination. 

The curves in Fig. 3 show hourly rates of CO2 uptake and of 
CO2 output in seeds which were briefly illuminated 2 h after 
imbibition. These rates are expressed as percentages of the gas 
exchanges in these seeds which were kept all the time in the 



SEED DORMANCY AND DORMANCY BREAKING 



187 





■ QCO2 [ 


QO2 


240 


/ 


■ 


220 


/ 


/ 


200 


/ 


/ 


180 


^ / 

A /^ / 


' / 


160 


/a' ^ ^^-t^ / 

A. A A ' ' A,A / 


/ 


140 
120 


/ A,i 

A '^^ 


A A A 

*■ /\ a-a' Va-a / 




y\/\j^ A-^r 


-^^6sX^ / ^^ 


100 


« r ^A. 


V^N/ \/ v^ 


y V * 


^ \ 




■ ■ ■ 1 U_J 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 L_l 1 


L i> 



4 8 12 15 20 24 

Hours 



8 12 16 20 24 



Fig. 3. QO2 and QCO2 of lettuce seeds (Grand Rapids). The straiglit line 
at 100 is the QO2 and QCO2 respectively in darkness and water, to which 
all the other values are related. QO2 and QCO2 respectively of seeds imbibed 
in water, which were illuminated for 30 seconds with 250 ft.c. of red light 
after 2 h of imbition (O — O). QO2 and QCO2 respectively of seeds imbibed 
in 10mg% dinitrophenol and illuminated (_!_ — _). QO2 and QCO2 
respectively of seeds imbibed in 10 mg°o dinitrophenol in the dark (▲ — A). 



dark. In illuminated seeds, imbibed in water, there is a small 
rise in rate during the first 12 h; this was found to be highly 
significant. The total amount of gases taken up or evolved during 
this 12-h period is higher, but not significantly so, in illuminated 
than in 'dark' seeds. This is because in discrete batches of 
illuminated seeds, the rate of gas exchange sometimes starts at 
a lower level and sometimes at a higher one than in 'dark' seeds. 
The main consideration is that respiration is affected in illumi- 
nated seeds. The difference in respiration between illuminated 
and non-illuminated seeds is seen in the tendency to rise of the 
exchange rate and not necessarily in the overall amount of gas 
exchange. It can, therefore, easily be overlooked, especially if 

References p. 190 



188 S. KLEIN 

only respiration at specific times is considered in different 
batches of seeds. 

This difference in respiration behaviour between 'Hght- 
treated' and 'dark' seeds may also be found in the presence of 
inhibitors. It is evident from the Figure that in the light-treated 
seeds, a 10 mg % dinitrophenol treatment results in a much 
stronger gas exchange than with 'dark' seeds, although germina- 
tion is inhibited in both cases. 

In the presence of coumarin, light has a similar tendency to 
increase respiration, and the tendency increases with the degree 
of inhibition of germination produced by the inhibitor. In 
general, light without doubt affects the respiratory apparatus 
during the germination period. 

It was mentioned earlier that seeds with their coats removed 
or pricked will germinate even in darkness. What about respira- 
tion in these? It turns out that their respiration during the first 
hours is much higher than in whole seeds. Thus, we again arrive 
at the possibility that gas exchange is limited by the coats and 
that all that light does is to render the endosperm more pene- 
trable to gases. Doubts have been expressed whether it is the 
actual removal or pricking of the endosperm which brings about 
germination, or whether the stimulation may not be due to the 
squeezing of the embryo itself during the process of coat 
removaF. Using deuteron irradiation with a controlled penetra- 
tion depth into the seed, it was possible to show that it is both 
necessary and sufficient to affect the endosperm alone in order 
to bring about germination^^' 21 (Fig. 4). Incidentally these 
experiments also showed that there is not necessarily a coiTela- 
tion between germination process and subsequent growth. A 
small dose of deep-penetrating deuterons bombarding the 
embryo itself leaves the endosperm unaffected, and therefore 
does not change the germination pattern, but causes a decrease 
in subsequent growth. On the other hand, a strong dose of 
deuterons applied to the endosperm alone will allow complete 
germination in darkness, without affecting the growth of the 
seedlings. This deuteron-induced germination cannot be reversed 



SEED DORMANCY AND DORMANCY BREAKING 



189 





EM (A) 
No effect 



(B) 

Medium increase 
in dark germination 
normal root length 




Strong increase 
in dark germination 
normal root length 






(D) 


(E) 


(F) 


Normal dark 


Strong increase 


Lethal 


germination 


in dark germination 


effect 


strong root 


strong root 




inhibition 


inhibition 





Fig. 4. The effects of deuteron irradiation on lettuce seed germination and 
subsequent root growth when applied to different depth levels. A to C: 
deuterons penetrating into endosperm only. D to F: deuterons penetrating 
deep into embryo. A and D: Weak dose: B and E: medium dose; C and 
F: strong dose. FC: Fruit coat; SC — EN: seed coat and endosperm; 
EM: embryo. The figures are not drawn to scale-". 



by Far Red irradiation, which means that a different mechanism 
is involved. 

This, however, does not mean that all the treatments we have 
mentioned affect the endosperm itself. For example, Koller^^ 
has shown that in certain plants the radicle itself is the light 
receptor, and recently good evidence in this respect has been 
brought forward for lettuce seeds by Ikuma and Thiman-^. 

Most probably, therefore, the chain of events starts in the 
embryo, possibly in the same cells which later on will be the first 
to grow and divide, but leads in the end to an effect on the 
endosperm, thereby resulting in increased gas exchange and 
perhaps reduced mechanical pressure on the embryo. As to what 
goes on in between these stages, our knowledge is less than 
scanty. It involves enzymatic activities which will be the subject 
of another lecture. Of course, I would be leaving this picture 
still more incomplete, were I not to mention that resumption of 
growth after a dormant stage certainly also involves changes in 



References p. 190 



190 S. KLEIN 

a very delicately balanced equilibrium between growth sub- 
stances and their specific inhibitors. This too will be discussed 
separately. Incomplete as this resume is, I hope it still shows 
that dormancy in seeds cannot possibly be dependent on one or 
a few substances only, or on one mechanism only, but that it is 
a state dependent on a large number of systems, which we are 
still unable to comprehend fully. 

REFERENCES 

1 D. KOLLER, Bull. Research Council Israel, 5D (1955) 58. 
" H. A. BoRTHwicK, S. B. Hendricks, M. W. Parker, E. M. Toole and 
V. K. Toole, Proc. Natl. Acad. Sci., Washington, 38 (1952) 662. 

3 M. Evenari, Symp. Soc. Exptl. Biol., II (1957) 21. 

4 S. Klein, Ph. D. Thesis, Hebrew University, Jerusalem, 1956. 

•5 N. C. Thornton, Contrihs. Boyce Thompson Inst., 8 (1936) 24. 

* W. Crocker and L. V. Barton, Chronica Botan. Camp. Waltham, Mass., 

1953. 
' H. E. TooLE, S. B. Hendricks, H. A. Borthwick and V. K. Toole, Ann. 

Rev. Plant Physiol., 7 (1956) 299. 
8 M. Evenari, Botan. Rev., 15 (1949) 153. 
^ H. R. Lerner, a. M. Mayer and M. Evenari, Physiol. Plantarum, 12 

(1959) 245. 
^0 M. Evenari, Encyclopedia of Plant Physiology, in press. 
11 H. A. Borthwick and W. W. Robbins, Hilgardia, 3 (1928) 275. 
1- M. Evenari, Radiation Biology, Vol. 3, (Ed. A. Hollaender), McGraw 

Hill Comp., New York, 1956, p. 518. 
1^ M, Evenari and G. Neumann, Bull. Research Council Israel, 3 (1953) 136. 
1-^ A. Kadman-Zahavi, Bull. Research Council Israel, 4 (1955) 370. 

15 A. Kadman-Zahavi, Ph. D. Thesis, Hebrew University, Jerusalem, 1959. 

16 G. E. Nutile, Plant Physiol., 20 (1945) 433. 

1" W. L. Butler, K. M. Morris, H. W. Siegelman and S. B. Hendricks, 

Proc. Natl. Acad. Sci., Washington, 45 (1959) 1703. 
1^ M. Evenari and G. Neumann, Palestine, J. Botany, Jerusalem Ser. 6 

(1953) 96. 
19 W. O. James, Plant Respiration, Clarendon Press, Oxford, 1953. 
^0 S. Klein and J. Preiss, Plant Physiol., 33 (1958) 321. 
21 J. Preiss and S. Klein, Plant Physiol., 33 (1958) 326. 
•" D. KoLLER, Ecology, 40 (1956) 20. 
-3 H. Ikuma and K. V. Thiman, Science, 130 (1959) 568. 



BIOCHEMICAL CHANGES IN BREAKING AND 
INDUCING DORMANCY IN SEEDS 

A. M. MAYER 

Department of Botany, Hebrew University, 
Jerusalem (Israel) 

The term dormancy is one which is used in various senses by 
various authors and this is particularly the case as regards the 
dormancy of seeds. In this paper, it is intended to use the term 
as referring to a population of seeds, specifically here lettuce 
seeds. If such seeds are placed under specified conditions of 
temperature and moisture, usually 26° in the dark, on moist 
filter paper, a certain number of them will germinate. The 
percentage of seeds which germinate under these conditions 
gives a measure of the dormancy of this seed population. This 
percentage can be altered in various ways, e.g., it can be in- 
creased or decreased by a number of physical and chemical 
agencies. Those which increase the percentage of germination 
may be considered as dormancy-breaking, while those which 
cause a decrease without, however, affecting irreversibly the 
eventual ability of the seeds to germinate when some other 
treatment is given, will be considered as inducing dormancy. 
From this it is immediately clear that, in referring to biochemical 
changes occurring during induction and breaking of dormancy, 
what is in fact being considered is the overall biochemical 
behaviour of a population of seeds under different treatments. 
In fact, the action of a dormancy-breaking and dormancy- 
inducing substance is always considered relative to the behavior 
of the untreated seeds. In no case is there any certainty that a 
specific change has occurred within any one given seed, and 
indeed, no attempt has ever been made to try and analyse 
individual seeds. This is for two reasons. 

First, lettuce seeds are extremely small and inconvenient to 
handle individually. The second and more important reason is 
that there is no way of foretelling whether any given seed will or 

References p. 198 



192 A. M. MAYER 

will not germinate without awaiting some indicative external 
sign, usually the protrusion of either the radicle or plumule from 
the seed coat, and by the time this has occurred germination has 
been completed. No other clear indication of whether germina- 
tion will or will not occur has been found for any seeds. Bio- 
chemical tests or histochemical staining have not proved ade- 
quate to answer this question. 

The two treatments of the seeds which we will now consider 
are using thiourea, which breaks dormancy, and coumarin, 
which is a germination inhibitor and in fact induces dormancy. 
The results to be considered, primarily those of Dr. Poljakoflf- 
Mayber and myself, are concerned with the biochemical changes 
which occur in lettuce seeds germinated in solutions of these two 
substances, as compared with those of seeds germinated in water. 
It may be said at the outset that in no case is there any certainty 
that these changes are in fact directly related to the dormancy- 
inducing or -breaking action of the chemicals concerned; in 
some cases, however, they are suggestive of this. 

During germination of seeds there is a general activation of 
all the enzyme systems in the seeds, due to water uptake. This 
activation is accompanied by a breakdown of certain storage 
products within the seeds, and by a general increase in the 
oxygen uptake. However, not all enzyme systems increase their 
activity at an equal rate and a few of them in fact rapidly 
decrease in activity as germination proceeds. Studies of the 
biochemical changes occurring in the germinating seeds have not 
yet been extended to all the metabolic processes that occur. 
Certain vital systems have not as yet been studied at all, whereas 
others have been shown to be of only secondary importance. It 
is therefore intended to confine the discussion to the breakdown 
of certain storage materials and to the possible oxidative path- 
way occurring during germination. 

It became clear fairly early in the investigation that in lettuce 
seeds the first substrate to be used during germination is the 
small amount of sucrose present^. The breakdown of this 
sucrose is not inhibited by either coumarin or thiourea during 



BIOCHEMICAL CHANGES IN SEED DORMANCY 193 

the early stages of germination. However, while in germinating 
seeds glucose appears as the sucrose disappears, this accumula- 
tion of glucose is completely blocked in seeds whose germination 
is prevented by coumarin (Table I). 

TABLE I 

THE EFFECT OF THIOUREA ON THE METABOLISM OF LETTUCE SEEDS AND SEEDLINGS 

Seeds germinated in thiourea (1250 p. p.m.) at 16' in the dark 

Metabolite Activity effect of thiourea Reference 

Sucrose utilisation Practically no effect 1 

Fat utilisation Slight inhibition 2,8 

Glucose accumulation Practically no effect 1 

Free fatty acid liberation Stimulation in later stages of growth 2 

Volatile fatty acids Slight stimulation 2 

Phytin breakdown No effect 6 



The principal storage material in lettuce seeds are lipids. The 
major decomposition of the lipids occurs at a fairly late stage of 
germination, but small amounts are probably already broken 
down at earlier stages. Both coumarin and thiourea cause 
rather extensive changes in the metabolism of the fats. Coumarin 
in general prevents the breakdown of lipids in the seeds-' ^ and 
at the same time the appearance of free fatty acids is also 
delayed or prevented while volatile ones increase. These latter 
need not necessarily and probably in fact do not arise from the 
lipids directly. 

The enzyme systems which are known to be active in the 
seeds in lipid breakdown are two lipases, one having maximal 
activity at acid pH and the other maximal activity at neutral 
pH. Neither of these enzymes show an increase in activity in 
seeds treated with coumarin (Table II). In contrast, thiourea 
inhibits the acid lipase, which in normal germination begins to 
increase in activity only at a fairly late stage of the germination 
process, while the neutral lipase activity rises initially and then 

References p. 198 



194 



A. M. MAYER 



TABLE II 

THE EFFECT OF COUMARIN //; VIVO AND /// vitl'O ON THE ENZYMATIC ACTIVITY 
OF VARIOUS HYDROLYTIC ENZYMES IN GERMINATING LETTUCE SEEDS 

In vivo: seeds germinated in coumarin solutions (100 p. p.m.). The enzyme 
assays were carried out in the absence of coumarin. 

In vitro: seeds germinated in water and coumarin was added to the reac- 
tion mixture. 



Enzyme 



Effect on activity 



in VIVO 



Effect on 
activity Reference 
in vitro 



Lipase, neutral Inhibits increase in activity 



Inhibits strongly 



Lipase, acid 
Phytase 



Activity remains as in dry seeds 

Inhibits increase in activity No effect 

Activity remains as in dry seeds 

Strong inhibition Slight inhibition 



3 
3 
6 



falls again in seeds germinated in thiourea^ (Table II, III). 
Despite these changes in the lipase itself the fat metabolism is 
far less profoundly affected by thiourea. Total breakdown is 



TABLE III 

THE EFFECT OF THIOUREA in vivO AND in vitrO ON THE ACTIVITY OF SOME 
HYDROLYTIC ENZYMES IN GERMINATING LETTUCE SEEDS 

In vivo: seeds germinated in thiourea (1250 p. p.m.). No thiourea present 

in the reaction mixture. 
In vitro: seeds germinated in water and thiourea added to the reaction 

mixture. 



Enzyme 



Effect on activity 
in vivo 



Effect on 
activity Reference 
in vitro 



Lipase, neutral Increase, then decrease 

Lipase, acid Inhibition progressing with time 

Phytase No effect 



Inhibition 3 

No effect 6 



BIOCHEMICAL CHANGES IN SEED DORMANCY 195 

hardly affected, free fatty acid formation is slightly depressed 
and volatile fatty acid formation is somewhat depressed. In this 
case, therefore, dormancy breaking with thiourea and dormancy 
induction with coumarin have opposite effects only on one part 
of the system, that is, the free volatile fatty acid. Although this 
fatty acid was not identified with certainty it seems probable 
that it is either acetic or lactic acid. In either case this would 
make it probable that its origin is closely connected with 
respiratory mechanisms. It seems more than doubtful in any 
case, that the changes observed are directly related to dormancy 
breaking or induction. 

Seeds which germjnate in the dark in water show an increasing 
oxygen uptake which starts almost immediately after the seeds 
are placed in the water. A number of respiratory enzymes are 
active even before the seeds are imbibed. These enzyme systems 
can be shown to exist if the dry seeds are extracted with water: 
the possibility that they become activated during extraction with 
buffer cannot be excluded. Those systems which have been 
shown to be active in the dry seeds, keeping the above limita- 
tions in mind, are a cytochrome oxidase, a number of dehydro- 
genases, a DPNH oxidase, catalase, peroxidase and a phenolase 
system, as well as what is possibly an ascorbic acid oxidase^' ^. 
Some of these enzymes increase greatly in activity as germination 
proceeds while others, particularly the glucose phosphate dehy- 
drogenase^o^ the phenolase and the DPNH oxidase^-^, do not, but 
even show a decrease. In this respect it is worthwhile to recall 
that during germination the number of cells in the seed or 
embryo increases. Therefore, the failure of an enzyme system to 
increase as germination proceeds implies that the average 
amount of enzyme per cell is decreasing. It is of course also 
quite possible that in some parts of the embryo the system does 
not develop at all while in others it maintains its original 
activity. 

It has been shown that during normal germination, the tri- 
carboxylic acid cycle activity of mitochondria prepared from 
the seeds is very low initially, and then increases as germination 

References p. 198 



196 



A. M. MAYER 



TABLE IV 

THE EFFECT OF THIOUREA ill VIVO AND in vitrO ON THE ACTIVITY OF OXIDATIVE 
ENZYMES IN GERMINATING LETTUCE SEED 

In vivo: seeds germinated in thiourea (1250 p. p.m.). No thiiourea present 

in the reaction mixture. 
In vitro: seeds germinated in water and thiourea was added to the reaction 

mixture. 



Enzyme 



Effect on activity 
in vivo 



Effect on activity 
in vitro 



Reference 



Ascorbic acid 


Inhibition 


Inhibition 


6 


oxidase 








Phenolase and 


Slight stimulation or 


Strong inhibition or 


4 


coupled 


inhibition depending 


no effect depending on 




oxidations 


on substrate 


substrate 




Catalase 


Inhibition 


No effect 


1 


Peroxidase 


Strong stimulation 


No effect 


9 


Dehydrogenases 


Very slight inhibition 


Practically no effect 


5 


DPNH oxidase 


Inhibition 


Inhibition 


15 


Lettuce 


Stimulates the develop- 


No effect 


13 


mitochondria 


ment of the citric acid 







cycle in very young 
seedlings but has no 
effect on the activity of 
mitochondria isolated 
from other seedlings 



proceeds-^' ^■-' ^^ (Table IV). This finding must be contrasted with 
what occurs in seeds whose dormancy is altered. 

Coumarin, generally speaking, retards or prevents the 
development of, or increase in activity of the above enzyme 
system. In no case has a direct effect of coumarin in any of the 
systems mentioned in vitro been found. This observation can be 
extended by noting that the entire glycolytic system functions 



BIOCHEMICAL CHANGES IN SEED DORMANCY 197 

normally in peas germinated in coumarin"- ^^ Thus it appears 
that coumarin does not act by directly suppressing som^e 
specific enzyme. There are, however, indications that it may act 
indirectly through its effect on release of inorganic phosphorus 
from phytin^ and also on ATP metabolism^^ and may thus in 
some way control respiration, or rather the energy supply to the 
seeds, by interfering with the rate-determining or controlling 
steps during oxidation of substrates. 

The changes in the oxidative system brought about by treat- 
ment of the seeds with thiourea are far more widespread. 
Poljakoff-Mayber and Evenari^^ showed that the rate of oxida- 
tion of tricarboxylic acid cycle intermediaries was much more 
rapid by mitochondria isolated from seeds germinated in 
water. Thus it seems that the tricarboxylic acid cycle enzymes 
become active much more quickly in the presence of thiourea. 
Preliminary results indicate similar effects for the cytochrome 
system., or at any rate for cytochrome oxidase. In contrast to 
these systems which become more rapidly active, a number of 
systems are inhibited. Oxidation of ascorbic acid and of DPNH 
by extracts of lettuce seeds is completely prevented when the 
seeds are germinated in thiourea. The systems responsible for 
this oxidation are situated in the soluble part of the cell^^' i^. 

The oxidation of phenolic substrates is altered by germination 
in thiourea (Table II). Extracts of seeds germinated in thiourea 
do not show any blackening even on prolonged standing. These 
extracts still contain an active phenolase which is not itself 
inhibited by thiourea^^. However, the oxidation of quinol, 
apparently by a coupled oxidation, is completely inhibited both 
by in vivo and in vitro treatment. 

It is possible that such coupled oxidations and also ascorbic 
acid oxidase and DPNH oxidase, normally mediate part of the 
electron transport in the seeds. There is at present little evidence 
to show that in such electron transport systems oxidative 
phosphorylation occurs. The disruption of such a system by 
thiourea, leading to the more rapid entry into operation of the 
normal Krebs cycle and cytochrome system as energy-providing 

References p. 198 



198 A. M. MAYER 

mechanisms, may therefore be possible. One action of thiourea 
in breaking dormancy may then be the following: Normally, in 
the initial stages of germination, the seeds are basing most of 
their oxidative processes on the oxidation of glucose-6-phos- 
phate, probably via the pentose shunt and their electron 
transport system is based on transport via ascorbic acid or 
phenols, with either a coupled oxidation with phenolase as the 
oxygen carrier, or by direct participation of ascorbic acid 
oxidase and DPNH oxidase. At a later stage of the germination 
process these systems are replaced by the tricarboxylic acid 
cycle, glycolysis and the cytochrome system for electron 
transport. If the seeds are treated with thiourea, this whole sei 
of changes is brought into operation with much greater rapidity, 
because some of the other systems are depressed, and thus, it is 
tempting to suggest that thiourea breaks dormancy by in- 
directly calling into operation more rapidly the energy-releasing 
processes in the seeds. 

To put forward this hypothesis at the present stage is some- 
what premature, as the evidence is far from conclusive: it is, 
however, a possible working hypothesis which can be tested 
experimentally. It is by no means suggested that these are the 
only changes which are induced by thiourea. That other changes 
occur is becoming clear today, and it is possible that in order to 
break dormancy, a number of biochemical events have to occur 
simultaneously. There is some evidence that the hormonal 
system is also altered by thiourea treatment. 

REFERENCES 

1 A. Poljakoff-Mayber, Palestine J. Botany, Jerusalem, Ser., 5 (1952) 180. 
~ A. Poljakoff-Mayber and A. M. Mayer, J. Exptl. Botany, 6 (1955) 28. 
3 D. RiMON, Bull. Research Council, Israel, 6D (1957) 53. 
'^ A. M. Mayer, Enzymologia, 16 (1954) 277. 

^ A. M. Mayer, A. Poljakoff-Mayber and W. Appleman, Physiol. Plan- 
tar urn, 70(1957) 1. 
« A. M. Mayer, Enzymologia, 19 (1958) 1. 
' A. M. Mayer, Proc. Xlth Intern. Botany Congr., II, 1959. 
^ A. Poljakoff-Mayber, Bull. Research Council Israel, 2 (1952) 239. 



BIOCHEMICAL CHANGES IN SEED DORMANCY 199 

9 A. Poljakoff-Mayber, Enzymologia, 16 (1953) 122. 

10 A. Poljakoff-Mayber and A. M. Mayer, Bull. Research Council Israel, 
6D (1958) 86. 

11 A. M. Mayer, unpublished results. 

12 A. Poljakoff-Mayber, unpublished results. 

1^ A. Poljakoff-Mayber and M. Ewenari, Physiol. Plantarum, 11 (1958) 84. 
I'l A. M. Mayer, Physiol. Plantarum, 11 (1958) 75. 
IS A. M. Mayer, Enzymologia, 20 (1959) 313. 

Note added in proof: Recent work has shown that ascorbic acid oxidase is 
absent from lettuce seed and ascorbic acid is oxidized by phenolase in a 
coupled oxidation (Stavy and Mayer, Bull. Research Council Israel, in 
press, 1961. 

DISCUSSION 

Halvorson Jr. : Your finding of a shift to the tricarboxylic 
acid cycle during germination is very interesting, as the opposite 
is the case with bacterial spores. Is the system at this point 
sensitive to antimycin A, particularly in the presence of thiourea? 
Is germination stimulated by Krebs cycle intermediates? 

Mayer: I regret to say that we have not tried the sensitivity of 
antimycin A. As to your second question, there is no clear 
stimulation of germination by Krebs cycle intermediates. 

Avi-Dor: I wanted to ask you about the increase of activity 
of the tricarboxylic acid cycle in the mitochondria, noted by you 
during germination. This increase might be caused by an 
increase in either the number or the size of the mitochondria, 
and not by an increase in enzymatic activity. 

Poljakoff-Mayber: We hope to be able to solve this problem 
with the help of electron microscopy, by observing both the 
number and the size of mitochondria in different stages of 
germination. In dried seeds the oxygen uptake per mg nitrogen 
is very low. It increases during the process of germination from 
2-5 //1/min/mg nitrogen to several hundreds of jul/mm/mg 
nitrogen. 

Kohn: I do not understand why light should have any effect 
on seeds, since they have to be in the ground before they 
germinate. Could it not be the effect of infrared or heat radia- 



200 A. M. MAYER 

tion? My second question: is a case known in plants, similar to 
that in insects, where the life history of one stage of development 
may affect the dormancy of the next stage? 

Klein: Some seeds will only germinate on the surface of the 
soil and need light for it. It is, however, not a heat effect, as a 
short illumination with weak red light will give this effect. The 
effect of white light preventing germination might be protective 
for seeds germinating underground. As to your second question, 
there are cases where the photoperiodic regime of the mother 
plant has an influence on the seeds. 

Koller: a case of the photoperiodic regime of the mother 
plant, affecting the dormancy of the seed has been reported by 
Lona on Amaranthus; seeds formed on long days were more 
dormant than seeds form.ed on shorter days. 

Hestrin : I would like to point out that botanists working in 
morphology describe plant structures in their minutest detail. 
Once, however, they deal with biochemistry, they are satisfied 
with effects caused by whole organisms. For example, increases 
and decreases in activity were noted and ascribed to mito- 
chondria in lettuce seeds, without any indication as to the 
origin of these mitochondria, the work being performed on a 
mixture of tissues. It seems to me that this biological work can 
be brought to a biochemical level by the use of single cells or of 
homogeneous tissue. Would unicellular algae or pollen grains 
be suitable in the study of the dormant state? 

I would also like to propose a hypothesis as to the mechanism 
responsible for induction and breaking of dormancy: suppose 
the cell contains in one compartment nutrients and in another 
enzymes. The breaking of dormancy would be the rupture of 
the separating wall, the induction of dormancy the erection of 
such a separating wall. 

Poljakoff-Mayber : The lettuce seed we have been working 
on consists of the embryo and tissues which are to a large 
extent degenerated. It follows that the mitochondria must have 
come from the embryo. Within the embryo the concentration 
of mitochondria may vary, but such observations would require 



BIOCHEMICAL CHANGES IN SEED DORMANCY 201 

larger seeds. I am doubtful whether pollen grain would be of 
help in this study, since they are too simple compared with 
seeds. We would miss all the elements of interrelations. 

Mayer: I would like to defend myself against some of the 
attacks made by Prof. Hestrin. I pointed out that we worked 
with mashed up tissues, and that this fact limited the value of 
our results. However, in this way we obtained indications which 
will enable us to determine the specific parts of the tissue 
involved. 

Halvorson: Is there any difference in the heat resistance of 
the seed before and after germination? 

Klein : I am afraid this has not been tested. 

Goldwasser: I would like to suggest the use of the fluorescent 
antibody technique to detect the site of enzyme formation, 
provided you can prepare these enzymes in a reasonably pure 
form. 

Koller: In regard to Prof. Hestrin's proposal for work on 
homogeneous tissues, I would like to suggest fern spores. They 
might be preferable to pollen grains, since they are activated by 
light in the Red-Far Red system. 

Nachmony : I would like to suggest the use of Bryophyta as 
suitable organisms for the biochemical study of these problems. 
In these plants dormancy can be induced and broken by 
photoperiodic treatment. The fact that almost every stage of the 
life cycle, including young sporelings, is able to react to the 
same treatment, may indicate that this ability is common to 
almost all cells, thus making the whole organism nearly homo- 
geneous in this respect. 



REST IN BUDS OF WOODY PLANTS* 

R. M. SAMISH 

Division of Pomology and Viticulture, Agricultural Research Station, 

Faculty of Agriculture, Hebrew University, 

Rehovot (Israel) 



Continuing our previous discussion, it seems desirable to 
define 'dormancy'. Dormancy in plants is a suspension of 
visible growth accompanied by a slowing down of the rate of 
metabolism. We should distinguish between two types of 
dormancy. One, quiescence, is caused by unfavourable external 
factors ; when these factors are removed growth is resumed. The 
second type of rest may be defined as a suspension of growth 
due to internal factors. Even if the plant is provided with 
favourable conditions it will not resume growth, unless some 
internal factor is changed or some block removed. 

Those of you who came here from Jerusalem enjoyed, no 
doubt, the profuse bloom of plum trees in the hills, while you 
may have noticed that the trees were still dormant in the coastal 
plain. This is typical of Israel, where after the usual warm winter, 
bloom in colder areas {i.e. at higher elevation and at greater 
distance from the coast) occurs earlier than in the coastal plain. 
Thus we see in Table I that the bloom of Santa Rosa plums at 
Rehovot near the sea shore was later by 10 days than the bloom 
at Jerusalem up in the mountains. These observations are not 
in agreement with general phenological concepts, according to 
which bloom occurs earlier in warmer spring weather. The 
general phenological rule holds true for Israel after exceptionally 
cold winters, as seen in the Table. Not only is the relative trend 
as regards time of blossoming in warm and cool areas reversed 
as compared with that after a warm winter (except for Rehovot), 
but the average date of bloom is advanced by almost one month. 



* Publication of the Agricultural Research Station, Rehovot, 1960 Series, 

No. 337-E. 



REST IN BUDS OF WOODY PLANTS 203 

TABLE I 

EFFECT OF CLIMATE ON DATE OF FULL BLOOM OF SANTA ROSA PLUMS 



Location 



Date of full bloom 



warm winter cold winter 



Jerusalem (mountain crest) March 31 March 10 

Affulah (central valley) April 3 March 8 

Mishmar H. (northern coastal valley) April 5 March 2 

Rehovot (southern coast) April 10 March 9 



Thus, a cold winter, a longer period of chilling, reverses the 
abnormal situation brought about by the warm winter, i.e. a 
certain period of chilling is required in order to terminate rest. 
I should like now to show you the experimental effect of 
chilling. Pelee^, working in our laboratory, placed resting Kelsey 
plum shoots for various periods into a refrigerator at 4°. As 
Table II shows, increased periods of chilling progressively 

TABLE II 

EFFECT OF DIFFERENT PERIODS OF CHILLING DURING REST ON THE AWAKENING 

OF KELSEY PLUM BUDS 

Chilling period Blossoming Open buds 

(days) date (% of total buds) 






April 13 


7 


7 


April 7 


15 


21 


March 24 


17 


56 


March 10 


29 



advanced the awakening of buds in spring. One week of chilling 
put forward bud opening by about a week, while chilling for 
56 days advanced wakening by as much as 33 days. Futhermore, 
chilling not only advanced the date of bud opening, but also 
increased the number of buds which opened. Seven per cent of 
buds opened on the control shoots; one week of chilling doubled 



References p. 208 



204 R. M. SAMISH 

the number, and about four times this number of buds sprouted 
after the maximum chilling period. 

In warm countries, insufficiency of chilling prolongs rest, 
delaying bloom and fruit maturation, and reducing the number 
of buds which open. Thus, both the photosynthetic area and the 
number of blossoms are affected. These phenomena, together 
with certain additional physiological disturbances caused by 
prolonged rest, constitute a serious economic factor with the 
result that certain crops cannot be grown economically in 
regions with warm winters. 

Horticulturists — as in our department — try to solve this 
problem by methods such as selecting and breeding varieties 
with low chilling requirements. The artificial breaking of rest 
represents an additional possibility. Johannsen- was the first to 
do so by means of anaesthesia. Molisch^ interrupted the rest 
period by immersing plants in a warm water bath. The physio- 
logical mechanism of this treatment was explained by Boresch'^ 
on the basis of anaerobiosis. For trees, we studied a number of 
sprays, the effects of which are described in Table III. On the 

TABLE III 

THE EFFECT OF MINERAL OILS AND CERTAIN DINITRO-COMPOUNDS ON THE 

AWAKENING OF DELICIOUS APPLE BUDS 



Spray material 




Open 


buds ( %) 


Unsprayed control 






12.5 


Mineral oil, med. heavy, 4%, U.M.R.* 


100 




17.7 




75 




19.5 




65 




30.5 


Dinitrocompounds** in med. oil U.M.R.* 


75 






2,4-dinitro-a-naphtol 






29.7 


3,5-dinitro-o-cresol 






28.7 


2,4-dinitro-6-cydohexylphenol 






25.2 



* U.M.R. = unsulphonatable residue = % saturated constituents. 

** Dinitrocompounds = concentrations equimolecular to dinitrocresol, 



1.5 % in the mineral oil. 



REST IN BUDS OF WOODY PLANTS 205 

untreated shoots of apple trees only somewhat over 12% of the 
buds opened. With a mineral oil spray, even when it was 
completely saturated, we obtained an increase in sprouting of 
about 50%, although we could not expect from a saturated oil 
any chemical action. It would seem probable that the inert oil 
film interferes with the oxygen supply of the cells, again a situa- 
tion leading towards anaerobiosis. We also investigated the 
effect of mineral oils with different degrees of unsaturation. Our 
results show a considerable increase of the rest-breaking action 
with an increasing proportion of the unsaturated compounds in 
the oil (lower U.M.R.). Furthermore, we dissolved dinitro- 
compounds in the mineral oil. All three which we tried — and 
dinitrophenol which we studied in another series of tests — 
increased the rest-breaking action of the spray, thus doubling 
the number of growing buds of the control. Today this dinitro- 
cresol-mineral oil spray^ is used widely by growers in Israel 
and in certain other subtropical countries. 

The physiological action of dinitrophenol has been reviewed 
by Simon^. Its uncoupling action is caused by inactivation of 
enzymes concerned with oxidative phosphorylation. Since so 
fundamental a process may affect cell metabolism at several 
points, we cannot point to a definite reaction bringing about the 
breaking of rest. But we do find a common denominator with 
previously mentioned methods of rest breaking: dinitro- 
compounds lead cell respiration into fermentative pathways. 
Bahgat' in the laboratory of Bennett broke the rest of pear 
shoots by holding them under nitrogen and found subsequently 
in their tissues both ethyl alcohol and acetaldehyde. When he 
treated dormant pear shoots with alcohol or acetaldehyde, he 
was able to break their rest. Thus, we see that products of 
anaerobic respiration are effective in terminating the rest period. 

The entry of buds into the resting stage is affected by the 
length of the daylight period, as originally shown by Garner 
and Allard^ and investigated further in the laboratories of 
Borthwick^, Wareing^o ^nd Nitsch^^. They found with very 
different woody plants, that short days induce rest, while long 

References p. 208 



206 R. M. SAMISH 

days prolong growth. During early rest the effect of short days 
can be reversed by long light periods, but later on, when rest 
becomes deeper, it cannot be reversed any more by this treat- 
ment. At this stage chiUing is required in order to break rest. 

The perceptor of the photoperiodic stimulus is the leaf. As a 
matter of fact, we can prolong growth even into winter by 
defoliation. On the other hand in spring, towards the end of 
rest, deciduous trees will normally not have any leaves. Under 
particularly favourable conditions part of their leaves may 
however be retained. We had occasion to observe that, in these 
cases, the buds in the axil of such leaves will start to grow earlier 
than buds the subtending leaves of which have dropped. Thus, 
some substance or substances are formed in the leaves which 
affect the rest of the adjoining bud. 

These phenomena are interpreted as due to a mechanism 
involving the interplay of auxins formed in light and inhibitors, 
some of which are destroyed by light, and most by cold. Before 
the application of paper chromatography to growth substances 
it was impossible to distinguish whether the disappaerance 
during rest of auxin activity was due to a decrease of 'free' auxin, 
as suggested by Bennett and Skoog^'- or, primarily, the difference 
between the activity of growth-promoting and inhibiting sub- 
stances, as we suggested^^. 

The first separations of these substances in woody plants were 
carried out by Hembergi^ on Fraxinus and by Spiegel^^ in our 
laboratory on Vitis. They showed that the concentration of 
inhibitors clearly reflected the state of the rest period. Thus, in 
the grape vine (Fig. 1), the inhibitors reached a pronounced 
peak by the end of December, dropping to zero about two weeks 
before bud burst. They also disappeared after rest had been 
broken by cold treatment. Furthermore, comparing the curves 
for two Vitis crosses, V. vinifera x V. rupestris 1202, which 
requires little chilling, and V. vinifera X V. ber/andieri 41-B, which 
requires severe chilling, we note that the general level of inhibitor 
concentration is proportional to the chilling requirement. Also, 
the inhibitor concentration in the V. rupestris cross reached zero 



REST IN BUDS OF WOODY PLANTS 



207 




XL XK I 

Date of sampling 



H Nil 
\ 



Fig. 1. Inhibitory activity in neutral fraction of ether extracts from grape 
buds of two Vitis crosses. Expressed as positive curvature of the coleoptile 

of Avena after SpiegeU^. = V. vinifera / V. berlandieri; 

= V. vinifera , : V. rupestris 



in Spring, about 70 days before the V. berlandieri cross. Since 
these investigations were pubHshed, similar resuhs have been 
obtained with a number of woody plants, maximum inhibitor 
concentration always being found during 'mid rest'. On the 
other hand, with most plants growth-promoter activity is 
gradually reduced during entry into rest, disappearing entirely 
in some species during 'mid rest' and reappearing again towards 
the end of the rest period. 

Considering growth-promoting substances, the activity of 
indoleacetic acid would seem to be very pronounced in most 
plants; indeed Spiegel considered it to be the only promoter in 
grape vine. In many plants indolethylacetate and indolepyruvic 
acid play an important part. Furthermore, the Rf values of a 
few unknown growth-promoters have been reported. As regards 
inhibitors, two substances have lately been identified, but there 
would seem to exist quite a number of additional ones. In buds 
of peach trees Hendershott and Walker^^ identified naringenin 
(trihydroxyflavanone) and Housley and Taylor^' revealed the 
mixture of substances making up Bennett-Clark's beta inhibitor, 

References p. 208 



208 R. M. SAMISH 

which seems to occur in many plants, as a number of ahphatic 
acids, unsaturated polyhydroxy fatty acids, azalaic acid and 
scopoletin. 

As more of these substances and their enzymatic mechanisms 
become known, we will start to tread on more solid ground, 
whereas today we are dealing with uncertain hypotheses. Up to 
the present, research has dealt separately with different plant 
organs, presuming different mechanisms. Wareing and Black^^ 
have, however, pointed out that apparently the same mecha- 
nisms act in the different organs of the same plant — obviously 
there exist morphological modifying factors. 

Permit me a concluding remark in connection with this 
particular gathering. I was very much impressed by the similarity 
of processes occurring in the diapause of insects and those 
which we find with buds of woody plants. Most of us working 
within relatively limited spheres of research know little about 
what is going on in disciplines as far removed as entomology 
from plant physiology. I think that this symposium will have 
made a considerable contribution by calling attention to those 
similarities. 



REFERENCES 

1 D. Pelee, M. Sc. Thesis, Hebrew University, Jerusalem-Rehovot, 1951. 

2 W. JoHANNSEN, Def. Kofh Danske Videpsk. Skrifft, 8 (1897) 276. 

3 H. MoLiscH, Sitzber. kgl. preuss. Akad. Wiss., 117 (1908) 87. 

4 K. BoRESCH, Biochem. Z., 202 (1928) 180. 

5 R. M. Samisch, /. Pomol. Hort. Sci., 21 (1945) 164. 

6 E. W. Simon, Biol. Rev., 28 (1953) 453. 

7 U. Bahgat, Ph. D. Thesis, Univ. California, Berkeley, 1931. 

8 W. ^. Garner and H. A. Allard, J. Agr. Research, 23 (1923) 871. 

9 R. J. Downs and H. A. Borthwick, Botan. Gaz., 117 (1956) 310. 

10 P. F. Wareing, Physiol. Plantarum, 3 (1950) 258. 

11 J. P. NiTSCH, Proc. Am. Soc. Hort. Sci., 70 (1957) 526. 

12 J. p. Bennett and F. Skoog, Plant Physiol., /i (1938) 219. 

13 R. M. Samish, Ann. Rev. Plant Physiol., 5 (1954) 183. 

14 T. Hemberg, Physiol. Plantarum, 11 (1958) 610. 

15 P. Spiegel, Bull. Research Council Israel, 4 (1954) 176. 



REST IN BUDS OF WOODY PLANTS 209 

16 C. H. Hendershott and D. R. Walker, Science, 130 (1959) 3378. 
1" S. HousLEY AND W. C. Taylor, J. Exptl. Botanv, 9 (1958) 458. 
18 P. F. Wareing and M. Black, The Physiology of Forest Trees, Ed. K.V. 
Thimann, Ronald Bros. Co., New York, 1958. 



DISCUSSION 

Koller: You mentioned artificial wakening of buds as the 
result of an increase in acetaldehyde or anaerobiosis in buds, 
what happens in this respect as a result of the cold treatment? 

Samish: It is thought that cold treatment destroys inhibitors. 

Avi-Dor: Do dinitrophenol and related compounds convert 
the aerobic respiration to glycolysis? Usually dinitrophenol 
increases respiration. Might it not be a lifting of the Pasteur 
effect? Is there any proof for an increase in glycolysis? 

Samish: This work has not been done with the type of 
material we are talking about, but it has been shown in animal 
tissues that energy-rich phosphorus bonds are not created and 
that respiration is directed into fermentative pathways. 

Avi-DoR : The alternative explanation for the lack of respira- 
tion might be the absence of an ATP acceptor. The addition of 
dinitrophenol breaks down ATP and respiration can start. 
There must not necessarily be a conversion from aerobiosis to 
anaerobiosis. 

Reinhold: I should like to point out in answer to Dr. Avi- 
Dor's earlier question that in both shoots and roots occurs a 
reversal of the Pasteur effect in the presence of dinitrophenol, 
with the occurrence both of anaerobic respiration and of 
increased glycolysis. 



LIBRAfiylpo 



ROUND TABLE DISCUSSION 

moderator: s. hestrin 

The 'Round Table' lasted for two sessions and only a few 
comments can be cited here. 

SESSION I 

Moderator (Hestrin): We have been called to the 'Round 
Table' under an injunction to seek for simple formulations of the 
dormancy problem, and to try to find common denominators 
in the mechanisms of cryptobiosis as they occur in widely 
divergent forms of life. It will be one of our tasks, no doubt, 
merely to determine whether indeed it is reasonable at all to 
seek for such common denominators. 

At first approach one is inclined to suspect that there may be 
at least two quite different classes of cryptobiosis which should 
be considered separately : (a) a transient class in which metabolic 
depression induced exogenously is promptly reversed when the 
environment is returned to normal; (b) a persistent class in 
which metabolic depression induced exogenously and/or en- 
dogenously, is not directly reversed on the reversion of the 
environment to normal. 

{I) Anhydrobiosis 

Let us first consider a kind of cryptobiosis, which is perhaps 
of the exogenous kind — the state of anhydrobiosis induced by 
lyophilization. Would Dr. Kohn care to recapitulate for us 
some of his conclusions in this regard? 

Kohn: In lyophilization you suspend metabolism by re- 
moving water. Most of my lecture dealt with the technical 
details of the method used in removing the water so as not to 
kill the organism in the process. This removal takes place in the 
frozen state, and one of our most important findings is that, 



ROUND TABLE DISCUSSION 211 

unless protected by special substances, the micro-organisms are 
killed in the dry state by oxygen. The hypothesis we proposed 
regarding the mode of the action of the oxygen invokes the 
paramagnetic properties of the molecule. Whether the hypo- 
thesis is valid in regard to micro-organisms other than those we 
have used, let alone other systems, remains to be learnt. 

Moderator: Do you consider, Dr. Lees, that given the proper 
techniques all forms of life could remain viable when desiccated, 
as is the case with micro-organisms? 

Lees: No, I think that most multicellular animals would 
succumb to complete desiccation. The explanation may have 
been provided by Prof. Halvorson when he emphasised the 
importance of structure in preserving viability. Desiccation 
leads to irreversible structural changes. 

Moderator: Should we perhaps assume then that there has 
been in certain organisms a specific evolution of a mechanism 
which prevents structural injury on desiccation? 

Lees: It would seem so. Although the extraordinary Chiro- 
nomid PoIypediJwn vanderphnki obviously shrinks on desicca- 
tion, it must have some means of preserving essential structures 
from damage. In this respect it is almost unique: other species 
of bloodworms die as soon as they have lost about three 
quarters of their body water. 

Halvorson : I would like to quote here the results of some 
experiments we performed in our laboratory some time ago. We 
measured the moisture affinity of some vegetative cells and in 
spores. This was done by measuring the equilibrium vapour 
pressure of the moisture in spores and in vegetative cells at 
different stages of drying. We found that in spores the vapour 
pressure remained close to that of pure water, until nearly all 
of the water had been removed, but in the vegetative cells the 
equilibrium vapour pressure fell rather rapidly while the 
moisture content was still relatively high. This would indicate 
that in the spore the water is free, that its removal does not 
affect the structure, and that there may be relatively little 
shrinking in a dry spore compared to a wet one. In vegetative 



212 moderator: s. hestrin 

cells, however, the amount of shrinking on drying is of the 
same order as the shrinking in a drying piece of meat. This 
would explain the sensitivity of non-frozen vegetative cells to 
drying, and serves as still another example of the importance to 
be ascribed to structure conservation for the preservation of 
viability during drying. 

(II) Resistance and Cryptobiosis 

Moderator: The endogenous class of cryptobiosis might 
perhaps be defined as a hypometabolic state whose sustained 
duration involves an endogenous regulatory mechanism. Would 
anybody like now to demolish this definition? 

Koller : I have no intention of tearing it down without some 
previous study. But you should note that a state of dormancy 
is one that enables an organism to pass through an unfavourable 
environment without drying. It seems to me that the important 
criterion is the resistance to external conditions. It is this which 
determines whether a state is dormant or not. 

Lees: I am afraid that this would not do for insects. If you 
take, say, resistance to cold, it is true that dormant insects are 
often resistant, but other insects, without a diapause, are some- 
times equally cold-hardy. 

Halvorson: As for micro-organisms, cells in a dormant state 
have an enhanced resistance to their environment. In bacterial 
spores we use loss of heat resistance as a means of determining 
whether dormancy has been broken. 

Samish : In the cells of a bud going into rest the protoplasm 
becomes more viscous and less transparent whilst the proportion 
of bound water is increased. Bennett in Berkeley found a 
correlation between the depth of rest and the amount of bound 
water. With the breaking of rest the protoplasm becomes more 
liquid and the bound water percentage is decreased. 

Halvorson : I was a member of the faculty of the University 
of Minnesota when Dr. Gortner first introduced the term: bound 
water. I then strongly disagreed with him, and I still think it is 



ROUND TABLE DISCUSSION 213 

a term that does not describe something that really exists. It can 
be found in the literature that the spore's resistance is increased 
because the water in the spore is bound. That is not so. Water in 
the spores is perfectly free. As I previously pointed out, one can 
evaporate almost all of the water of the spores and the vapour 
pressure will remain that of pure water. 

Lees: I might perhaps add that certain species of insects are 
often found to be more cold-resistant in the dormant condition 
than when actively growing and developing. However, as 
R. W. Salt has shown, this may merely be a consequence of the 
presence of 'foreign' particles in the gut of the feeding insect. 
The physiological mechanisms responsible for dormancy and 
cold-hardiness may well be entirely different. We tend to think 
of insect diapause primarily as a growth phenomenon. 

Mandelbaum : We should also note that in trees frost- 
resistance and dormancy can be induced quite separately. 

Moderator: Let us now ask the protozoologists and myco- 
logists whether they think that resistance and metabolic dor- 
mancy are separable? 

Wahl: Yes, indeed. In fungi they are entirely separable. 

Moderator: We have decided, I think, that hypometabohc 
dormancy and enhanced resistance are separable phenomena 
though in some systems they are correlated. 

Mayer: The correlation might arise as follows: a dormant 
organism has a lower requirement for energy-releasing pro- 
cesses. Therefore it is less affected by external factors. In the 
growing organism, on the other hand, there is a much higher 
requirement for energy-releasing processes. Hence external 
factors affect a growing cell more readily. 

(Ill) Metabolism in Cryptobiosis 

Moderator: This brings us now to an important further 
question — whether the hypometabohc state is characterised by 
a quahtative change from normal in the metabolic pathways. 

Avi-Dor: I would like to call vour attention to the fact that 



214 moderator: s. hestrin 

respiration in the hypometabolic state depends largely on 
systems which are insensitive to CO and narcotics. Such systems 
are not linked to the cytochromes, are not phosphorylating and 
perhaps do not yield much energy in a useful form. The assump- 
tion could thus be made perhaps that in the hypometabolic 
state the organism can afford to use a system which is energeti- 
cally less effective but which is less sensitive to inhibition. 

Lees : Investigators who have studied diapause in the Cecropia 
silkmoth have reached a different conclusion. Williams, 
Shappirio and Schneiderman are now of the opinion that the 
dormant state is not accompanied by any qualitative change in 
the metabolic pathway, only by an alteration in the quantitative 
relationships of the terminal enzyme, cytochrome oxidase, and 
cytochrome c. CN- and CO-resistance in the diapausing pupa 
is thought to be the result of the virtual disappearance of cyto- 
chrome c and the great excess of oxidase. This system, which 
would also be less effective energetically, would permit the 
stored reserves of fat and glycogen to be used sparingly. 

Halvorson: From what we know of the enzyme pattern of 
spores, it appears that while those enzymes needed for energy 
are preserved, a number of enzymes associated with biosynthetic 
reactions are acquired during outgrowth. Thus different enzyme 
patterns would be associated with the formation, maintenance 
and outgrowth from the dormant state. 

Lees: Is it not possible that the maintenance respiration 
could be sustained by changes in the classical cytochrome 
system? 

Avi-Dor: There is really no fundamental difference between 
the two views. The electron transport system might be the same 
up to a point, but if cytochrome c were missing, the final 
electron acceptor the reduced flavin proteins would have to be 
reoxidized directly by oxygen. Thus the pathway would still be 
largely the same but would break off at another point. 

Poljakoff-Mayber : Perhaps what is involved here is not so 
much a complete switchover as a change in proportion between 
the various pathways. I wonder whether there is not also a 



ROUND TABLE DISCUSSION 215 

Structural aspect to be considered. The various components of 
a system can of course only interact if they concur in time and 
within a narrow space. 

Halvorson Jr. : In answer to Dr. Poljakoff-Mayber, we have 
recently found mitochondrial type particles in vegetative cells 
of Bacillus. These particles were also present in the spores but 
they lacked a number of enzymes associated with cytochrome 
systems. The incomplete particles constitute a functional differ- 
ence between spores and vegetative cells. I am rather inchned to 
believe that the differences between spores and sporulating cells 
must be very subtle, in view of the great similarity between a 
system going into the dormant state and the dormant state itself. 

Lees: 1 think I mentioned in my lecture that mitochondria 
which are readily visible in the developing embryo of grass- 
hoppers, 'disappear' when the embryo goes into diapause. They 
reappear when it comes out of diapause. 

Mayer: May I enquire what exactly is meant here by dis- 
appearance and reappearance? 

Lees: My remark was based on the work of Bucklin and his 
associates who have stained the mitochondria with Janus green 
B and examined the preparations with the light microscope. 
Actually, mitochondria are present during diapause but take the 
stain very lightly, indicating that there is a probable deficiency 
in the cytochrome system. 

Halvorson Jr. : In spores one observes under the electron 
microscope particles very similar to those in the vegetative cell. 
The mitochondria-like particle in the spore is non-functional. It 
contains several of the enzymes but does not contain a complete 
oxygen transport system. In the vegetative cell there is a small 
quantity of the soluble DPNH oxidase. In the spore its quantity 
is very high and accounts for all of the passage to molecular 
oxygen. So you employ alternate systems as you go from the 
spore to the vegetative cell, or the other way. 

Mayer: In seeds, too, one suspects such a switch in electron 
transport systems with dormancy. It is very pleasing to hear of 
an even clearer case of it in the bacterial spore. 



216 moderator: s. hestrin 

Keynan: Does not a change of this sort in fact also occur in 
insects? 

Mayer: From Dr. Lees' comments it seems that as far as 
insects are concerned the verdict should be a 'not proven' 
rather than a 'nonexistant'. It seems to me that by the use of 
the Britton-Chance spectrophotometer technique we could learn 
whether or not there is a change in cytochrome Z?5 and cyto- 
chrome c in diapause, and thus show whether or not the 
cytochrome system is operative in this stage. 

Lees: A dramatic fall in cytochrome c concentration has in 
fact been demonstrated by Shappirio using low temperature 
spectroscopy. 

Poljakoff-Mayber : We cannot be sure that there is no 
previously accumulated energy-store in the form of ATP. This 
store could be used during dormancy. After all, in the latent 
state of metabolism there may not be much need for energy. 

Halvorson Jr.: We do not know the ATP/ADP ratios in 
the spore. It would be worthwhile indeed to measure this. 
There are only FitzJames' observations on this subject, on a 
correlation between germination and polyphosphate degrada- 
tion. 

Mayer: The polyphosphate, phytin, might be such an energy 
store in a dormant seed. 

Moderator: The difficulties of the approach to this problem 
are formidable. I would not wish to sound pessimistic, but 
please realize that whereas in the bacterial system we know what 
cell is dormant, and can test that cell, in insects and plants there 
appears to be as yet no accurate location of dormancy, and no 
means therefore of differentially examining the critical locus. 

Samish : Well, in buds, some think that the scales form a wall 
which prevents the entry of oxygen and thus induces dormancy, 
but others hold the opposite view, that it is the dormancy which 
induces the formation of an impermeable protoplasmic mem- 
brane. A group of Russian workers has correlated entry into 
dormancy with constriction of the protoplast and resultant 
scission of the plasmodesma. They have also found that when 



ROUND TABLE DISCUSSION 217 

there is less resistance to cold there is less tendency to the 
breaking of the plasmodesma. 

Lees: It depends on what you regard as the critical locus. The 
organ systems controlHng diapause in insect eggs, larvae and 
pupae are different. However, although diapause mechanisms, 
even in closely related species, have probably been evolved 
independently, natural selection has had to act on existing 
growth-controlling systems, such as the endocrine system. 
Therefore a certain uniformity emerges when the mechanisms of 
particular stages are compared. Some further similarities, 
especially in connection with respiratory metabolism, appear 
if the reacting tissues are regarded as the critical locus. 

Halvorson: In micro-organisms, those most resistant to the 
environment are spherical. Spores, too are all more or less 
spherical. I do not know though whether this is in any way 
significant. 

Moderator: It might be rewarding to focus our discussion 
upon the surface of the dormant organism. Is it not true that 
such a surface is generally waxy or at any rate hydrophobic? 

Lees: Well, as most insects possess a waterproof cuticle 
throughout development one would have to find out whether 
the waterproofing was more efficient during diapause. I do not 
think that this has yet been studied critically. It is perhaps worth 
mentioning, however, that silkworm pupae show a visible 
accumulation of wax after they have remained dormant for a 
year or two. 

Wahl: a morphological feature common to many fungi in 
the dormant stage is the thick wall which reduces penetration. 

Halvorson : It is true that spore cell walls are different from 
the vegetative cell walls. It is also probably true that Gram 
positive organisms, which are more resistant have a cell wall 
differing from that of the less resistant organisms. There may 
be a correlation between the type and composition of the cell 
wall and the dormancy and resistance of the cell. 



218 moderator: s. hestrin 

(IV) hiduction of Cryptobiosis 

Moderator: Perhaps if we make a list of elements which 
induce or break dormancy in different organisms we might find 
a few common denominators for the different systems. 

Halvorson : Dormancy in micro-organisms was once thought 
to be induced by unfavourable conditions. This is not so. But I 
think it is true that the inducement to form spores is occasioned 
by a change in the nutritional environment, and this perhaps 
may be true in other systems as well. 

Koller: The basic similarity in the various dormancy- 
inducing and -breaking mechanisms is the fact that they are all 
triggered by the environmxcnt. 

Lees: I agree. We have factors like dryness and lack of food 
as well as guide factors such as photoperiod. The response to 
the latter allows anticipation of unfavourable conditions. The 
dormancy-controlling factors are remarkably similar in buds 
and insects. In both cases induction is by photoperiod and 
breaking by chilling. But how far does this similarity really go? 
The guide stimuli are shared, but different response mechanisms 
have been evolved. 

Moderator: It is a credo of biochemists that there must be 
some common mechanism. We can only discover it by looking 
'under the skin'. The zoologist is trained to see many differences 
and dissimilarities. Specifically one asks whether the several 
photoreceptive and 'endogenous clock' mechanisms encountered 
in different species are not in principle similar? We know so 
little, it is perhaps better not to try to answer this particular 
question yet. We are now concluding our discussion for today, 
but I would very much like you to give some thought to the 
following questions by way of preparation for our second 
session. Is there perhaps an important experiment which you 
would like to see performed in this field? If so, would you like 
to describe it? Secondly, could you point to a biological system 
particularly suitable to the study of cryptobiosis? Can you 
suggest quantitative procedures which would facilitate a more 
exact approach to our problems? 



ROUND TABLE DISCUSSION 219 

Finally, would you care to say whether cryptobiotic states 
are a useful feature in evolution? 

SESSION II 

(V) The Selection of Suitable Biological Materials 

Moderator (Hestrin): A proper choice of biological material 
for the examination of the problems of dormancy could no 
doubt be a matter of critical importance for the advancement of 
knowledge in this area. Could we perhaps arrive at some 
agreement as to the kind of properties we would want in such 
biological objects of our choice? Perhaps Prof. Halvorson who 
has done so much of the pioneer work on this problem would 
consent to give us his opinion as to the manner in which the 
choice objects for studies should now be made? 

Halvorson: Little progress was made in the study of the 
physiology of the bacterial spore until it was realized that proper 
separation of the dormant stage (ordinary spore) was essential. 
I am sure this is applicable to all the other fields. 

Moderator: I suppose heterogeneity in the bacterial spore 
population might be a serious obstacle to quantitative studies 
of the phenomena. Is it now possible to get synchronized 
germination in homogeneous spore samples? 

Halvorson Jr. : I think that variations in time of germination 
result from variations in time of storage of the spores. As 
activated forms of spores show considerable loss of weight, we 
have been able to perform separation by equilibrium sedimenta- 
tion in the density gradient. Another heterogeneity was in 
resistance to ethylene oxide resulting from phenotypic variation 
in fat content against a constant genetic background. Here we 
accomplished a separation by electrophoresis. Thus by use of 
appropriate physical techniques one can get greater population 
homogeneity. 

Kohn: Would density gradient centrifugation ensure homo- 
geneity in spores? 

Halvorson Jr. : We know we can separate active spores from 



220 moderator: s. hestrin 

dormant ones by this method, and also heat-shocked spores 
from dormant spores. By refining the density differences, one 
should be able to separate spores with varying degrees of 
dormancy. 

Moderator: As a bystander one has the impression that 
microbiologists are fully aware of this aspect of the problem and 
that they are directing adequate efforts to the working up of 
reliable assay procedures. This may not be equally true, how- 
ever, in relation to some of the more compHcated biological 
systems. Would you like to comment. Dr. Lees, on good quanti- 
tative techniques for work with insects. 

Lees: The selection of the most suitable and the most stand- 
ardized material for insect dormancy studies has not really been 
a problem. You choose your insect according to your purpose ; 
for endocrine system studies you choose a large insect, like a 
giant silkmoth. For studies on, say, photoperiod, a quickly 
multiplying species is preferable. Unfortunately, such an insect 
is usually an inconveniently small subject for surgery. Lepidop- 
tera of moderate size have nevertheless been used rather exten- 
sively for both purposes. The silkworm of commerce, with its 
different genetic races, has been a particularly favoured species. 

Moderator: Might it not be useful to concentrate within a 
given insect on one particularly suitable tissue? 

Lees: If you mean a tissue suitable both for biochemical work 
and for the study of cell structure during dormancy, the epider- 
mis should prove to be a good choice. It forms a fiat layer only 
one cell thick and is easily stained and preserved as a whole 
mount. Wigglesworth has shown that when moulting is induced 
by injection of ecdyson, detectable cytological and cytochemical 
changes occur within 24 h. Shappirio has also used wing epithe- 
lium for in yivo spectroscopic studies. 

Moderator: Could one cut this tissue up and put it in a test 
tube? 

Lees : The epidermis could certainly be separated and studied 
biochemically. But if you are referring to the possibiUty of 
tissue culture, the prospects are not bright. As for organ culture, 



ROUND TABLE DISCUSSION 221 

media are known in which lengthy survival is possible, but there 
is usually no growth. 

Harpaz: We were recently contemplating an extraction of 
haemolymph from insects with a view of separating the haemo- 
lymph cells and growing them in culture. Should this succeed 
we might also have a suitable material for dormancy studies. 
Some of these cells are fairly large and can be studied with the 
ordinary light microscope. We could get about a couple of 
milliliters of material from a fairly large insect. 

Kindler: Bearing in mind Prof. Shulov's suggestion that lack 
of movement is a sign of dormancy in insects, might it not be a 
good idea to use muscle tissue? 

Lees: There would be no technical difficulty. You could 
dissect the muscle tissue free from anything else, except hemo- 
cytes from the blood. However, the muscle tissue of an insect 
in diapause may not show any characteristics peculiar to the 
diapause condition. In the Cecropia silkworm, the muscles 
contain their full complement of enzymes and are still fully 
functional; thus the insect is able to wriggle its abdomen. 

Galun: Even if we did succeed in getting a tissue culture, it 
would be no benefit for the study of this aspect of dormancy. 
The concept of diapause does not apply to the cells once they 
have been removed from the regulatory system of the body. 

Moderator : Perhaps we should not take so extreme a view. It 
should be remembered that in the mammalian system it was for 
a long time generally thought that no hormone effect could 
operate outside the intact organism. Yet this is no longer so. 

Galun: No, I assume some hormone effect could be obtained, 
but not in the case of arrested growth. 

Moderator: What is the position in this regard as to fungi? 

Wahl: I am afraid that it will be extremely difficult to find 
among fungi genetically pure and uniform material for dor- 
mancy studies. 

Nachmony: a liverwort which I have been working with 
might be a particularly suitable test object. In this organism, one 



222 moderator: s. hestrin 

can induce dormancy by 16 long days, and break it by 3 short 
days. All the stages of the life cycle manifest this response 
pattern. I tried it out both on the whole gametophyte and on 
the thallus which I had cut into pieces, and obtained the same 
results in both. Another advantage of this material is that the 
gemmae — the vegetative reproductive organs are genetically 
uniform. Dormancy can be induced in them in the gemmacurp. 
One can thus obtain at will either dormant or nondormant 
gemmae. We have so far observed these phenomena but know 
very little about the biochemical behaviour. The suitable 
temperature for growing this material is about 15° and the 
required light intensity about 120 ft.-c. 

Moderator: This seems to be truly a most attractive and 
uniquely suitable biological material for the study of dormancy. 



(VI) Memory Phenomena in Dormancy 

Moderator: Several of the speakers have pointed to the 
operation of the memory phenomena in relation to induction 
and duration of dormancy in different systems. What might be 
the physical basis of these effects? 

Lees: The mechanism controlling diapause termination 
provides a kind of memory, usually of past temperatures which 
are integrated in a very particular way. As a model system we 
could suppose that two chemical reactions are competing for a 
given substrate; or, simply, that there are opposing synthetic 
and breakdown reactions. In both cases the reaction must have 
different temperature coefficients. As yet we have no inkling as 
to what these hypothetical reactions might be. 

The neurosecretory cells are involved in another type of 
memory when, in diapause induction, they 'record' the photo- 
periodic stimulus even though its effect is not manifested until 
much later in development. The photoperiodic measurement of 
time itself involves a third kind of memory, the nature of which 
is still largely obscure. 



ROUND TABLE DISCUSSION 223 

(VII) Proposals for Future Work 

Moderator: Let us consider finally experiments still to be 
performed on the induction and the breaking of dormancy in 
the different biological classes. 

Halvorson Jr. : A simple explanation for many dormancies 
is that they are simply expressions of an anhydrous condition. 
A way of investigating this might be the exposure of a system to 
deuteriated or tritiated water. Then after rupturing the tissue in 
a non-aqueous solvent, we could look for deuterium or tritium 
on some recognisable internal component. One would then find 
whether water could enter such a component. The other question 
is whether there is any metabolism in the anhydrous system. We 
might be able to decide this point by determining whether there 
is incorporation of ^'-P into ATP within the 'anhydrous' cell. 

Jashphe: It might be very important to determine whether 
variations in the permeation barrier accompany the induction 
and breaking of dormancy. 

Halvorson: I agree this is very important, but the design of 
a suitable experiment on this is a very difficult problem. 

Mayer: In bacterial spores, it is very often a heat shock that 
breaks dormancy. In seeds too, a heat shock of short duration 
and at moderately high temperature performs the same function. 
All this seems to point to some change in the physical structure. 
However, I have no idea how to establish what this structure is. 

Wahl: I have two suggestions. One is based on the obser- 
vation that when we inoculate oat seedlings in one-leaf stage 
with uredospores of race 2 of Puccinia graminis avenae, uredia 
are produced on the leaf and teHa are formed on the coleoptyle. 
In other words, the same inoculum produced the active stage 
of the rust fungus on the leaf and the dormant stage on the 
coleoptyle. The question arises why does a similar genetic stock 
yield on the same plant dormant and non-dormant spores, 
depending only on the kind of tissue used as a host. The second 
proposal stems from Yarwood's studies on the powdery mildew 
of red clover, Erysiphe polygoni. Conidiospores collected during 



224 moderator: s. hestrin 

the day germinate readily while those collected at night 
germinate poorly or not at all. 

The mechanism underlying these phenomena deserves further 
elucidation. 

Halvorson: I have no pet experiment to suggest, but I do 
want to make a general suggestion, applicable perhaps to all the 
fields. It is generally agreed that the study of bacterial spores 
has arrived at a rather ideal technique. The reason for this is, 
that the investigations were centered for a Ions time on the 
initial changes rather than on the phase of outgrowth. In doing 
so only germination triggering was studied. Therefore some 
similar techniques would have to be found in the other fields 
before some real progress could be registered. 

(VIII) Purpose of Dormancy 

Moderator: There is one further aspect we can still touch on 
briefly: the purpose of dormancy. This is of course teleological, 
but I think this is permissible on the last day of the conference. 
Do we have dormant stages which are useful as distinct from 
resistant stages? 

Halvorson Jr. : I would like to quote some work done by 
Dr. Knight and his colleagues at Wisconsin University on the 
enzymatic oxidation of sterols. They found that this reaction 
takes place only in the spore and is absent in vegetative cells. 
The presence of specific antigens in bacterial spores, of cysteine- 
rich structures as shown by Vinter, of heat-resistant enzymes and 
of compounds such as dipicolinic acid, all attest to distinctive 
features of the dormant state in bacterial endospores. 

Lees: I accept the invitation to become teleological! I have 
emphasized that the diapause state does not necessarily go hand 
in hand with resistance to adverse environmental factors such 
as low temperature and water lack. Diapause should rather be 
regarded as a timing device which ensures that the actively 
developing feeding stages appear at a time when these factors 
are favourable. When this delicate adjustment is upset by 



ROUND TABLE DISCUSSION 225 

removing the insect from its natural environment, dramatic 
consequences may follow. I have been told that the Cynthia 
silkmoth was once quite common in New York where it 
produced two broods a year, as it did in its native South East 
Asia. In New York, however, average temperatures are often 
higher and in one particularly hot summer, a third generation 
was begun which failed to reach completion before the Aikmthus 
trees lost their leaves in the fall. The resulting population crash 
has remained a permanency. 

Moderator: We have now come to the end of our discussion. 
I would like to thank all of you for your active participation. 
I am wondering what the position will be in a few years when 
we meet again to discuss cryptobiosis. I believe the whole 
classification of the subject will be different and perhaps Dr. 
Keynan might consider the reconvening of this conference in a 
few years. 

On behalf of all of you, I would like to thank him for the 
hospitality he has extended to us. I would also like to thank 
most warmly our guests the Halvorsons — father and son, and 
Dr. Lees for their most stimulating contributions to our dis- 
cussions, and finally all of you for the friendly and scientific 
spirit in which our work was conducted. 



SUBJECT INDEX 



Abiosis, 1 

Abraxas niiranda, 127 
Acantocephala, 101 
Acanthor, 101 
Acetic acid, 42 
Acetylcholine, 137 
c/5-Aconitic acid, 46 
Acronycta rumicis, 128 
Adenosine, 32, 33, 79 
Adenosine deaminase, 79 
Adenosine triphosphate, 79 
Adipic acid, 46 
Adventitious buds, 35 
Aestivation, 104 
After-ripening of seeds, 13 
Ageing of bacterial spores, 13, 34, 

64 
Alanine racemase, 36, 75, 86 
D-Alanine, 36 
— , inhibitor of L-alanine activation 

of bacterial germination, 68 
L-Alanine, 32, 33, 45, 64, 79 
— , activation, 68 
— , mechanism of utilization, 85 
— , nature of initial binding site for, 

87 
P-Alanine, 89, 91 
L-Alanine dehydrogenase, 64, 79, 

87, 89, 91 
Albumin, 27 
Aldolase 79 
Alternaria so/ani, 113 
Amaranthus retroflexus, 180 
Ametabolism, 1 
L-Aminooxidase, 69 
a-Aminopimelic acid, 46 
Anguina tritici, 100 
Anabiosis, 1 

Anhydrobiosis, 15, 210-212 
Anopheles macu/ipennis, 128 
Anoxybiosis, 15 



Arginine, 45 

Arsenite, 80, 91 

Ascaris, 98, 99 

Asparagine, 45 

— , effect on inhibition of bacterial 
sporulation, 47 

Aspartic acid, 45 

— , effect on inhibition of bacterial 
sporulation, 47 

Aspergillus niger, 113 

Atabrine, 84 

Australorbis glabratus, 105 

Austroicetes cruciata, 144 

Azelaic acid, 208 

Bacillus cereus, 33 

— , time of sporulation, 41, 42 

— , germinating agents of, 78 

— , growth medium, 47 

Bacillus licheniformis, 64 

Bacillus megatherium, 33 

— , germination of spores, 77 

— , release of dipicolinic acid from, 
38 

Bacillus stearofhermophilus, 33 

Bacillus subtilis, cytochrome con- 
tent, 16 

— , production of sclerotia, 116 

Benzaldehyde, 110 

Biotin, 114 

Bivalent cations, level in bacterial 
spores, 72 

Bulinus truncatus, 105 

Bloom of woody plants, 202 

Bombyx mori, 122 

Botrytis cinerea, 1 1 1 

Calcium, 48 

— , effect on germination of bac- 
terial spores, 34, 35 

— , effect on dipicolinic acid syn- 
thesis, 74 

— , level in bacterial spores, 72 



SUBJECT INDEX 



227 



Calcium chloride. 68 

Calliptamiis palaestinensis, 146 

Capillar ia hepatica, 97 

Carpocapsa pomonella, 124 

Catalase. 36, 75 

— , in dry seeds, 195 

Cercaria, 99 

Cestodes, 99, 101 

Chemical nature of the dormant 

state of bacterial spores, 71 
Chilling, 203 
— , effect on termination of rest in 

woody plants. 204 
Chlamydospores, 109 
Chloramphenicol, 18 
Chloroform, 110 
Chlorohydrin, 8 
C/wrthippus brunneiis, 150 
Citellus citellus, 105 
Citric acid, 46 
Citrulline, 45 

Clostridium roseimi, 39, 40 
Clostridium acetobutylicum, 76 
Cobalt, inhibitor of bacterial spore 

germination, 34 
— , effect on inhibition of sporula- 

tion, 48 
Cocarboxylase, 80 
Colletrotrichum lindemuthianum. 1 1 1 
Copper, 48 
Coracidium, 99 
Corpus allatum. 133 
Corpus cardiacum, 132 
o-Coumaric acid, 117 
Coumarin, 117 
— , germination inhibitor of seeds, 

169, 184, 185, 188. 192, 196 
— , effect on metabolism of fats in 

seeds, 185, 188, 193 
— , stimulation of germination of 

uredospores, 154 
Cryobiosis, 15 
Cryptobiosis. bacteria. 11, 15 



— , cysts of protozoa, 9 

— , fungi, 107 

— , induction of, 218 

— , insects, 10, 120 

— , metabolism in, 213-217 

— , multicellular resting bodies, 113 

— , plants, 159 

— , parasitic worms, 97 

— and resistance, 212, 213 

Cydia pomonella, 1 54 

Cysteamine. 33 

Cysteine, 23. 45. 88 

Cystine, 45 

Cytochrome, 128 

Cytochrome oxidase, 138 

— , in dry seeds, 195 

Dactylogyrus vastator, 98 

Dauereier, 98 

Dauerlarve, 100 

Decoy plants. 108 

Dehydrated forms, 2 

Dehydrobiosis in seeds. 5 

Dehydrogenase in dry seeds, 195 

Deuteron irradiation, 188 

Diaminopimelic acid. 45, 60 

Diapause, 120, 154 

— , biochemistry, 132 

— , endocrinology, 132 

— , facultative, 123 

— , geographical variations in, 127 

— , induction of, 121 

— , mode of action of environmen- 
tal factors on, 135 

— . nutrition in, 124, 136, 156 

— , obligatory, 123 

— , population density factor. 124 

— . temperature, 123, 125, 133, 137, 
156 

— , termination, 124, 136 

Diaphorase, 83 

Diethyl L-glutamate, 50 

— , malonate, 49 

— , oxalacetate, 49 



228 



SUBJECT INDEX 



— , pimelate, 55 

— , succinate, 49 

Dihydropicolinic acid, 85 

Diketopimelic acid, 85 

Dipicolinic acid, 32 

— , block of synthesis by ethyl oxa- 
mate, 50 

— , effect on germination, 32, 35 

— , effect on production of heat sen- 
sitive spores, 58 

— , effect on viability and heat resis- 
tance of spores, 73 

— , level in bacterial spores, 37, 73 

— , stimulation of ATPase, 82 

— , stimulation of DPNH oxidase, 
84 

— , stimulation of glucose oxida- 
tion, 73, 81 

— , pathway of synthesis, 85 

Dinitrocresol, 8 

Dinitrophenol, 18, 139, 184, 185, 
188 

Diplogaster, 100 

Dociostainus maroccanus, 144, 146 

Dormancy, 1, 175 

— , buds, 7, 202 

— , bacterial endospore, 71 

— , insects, 121 

— , seeds, 5, 159 

— , memory phenomena in, 222, 223 

— , purpose of, 225 

— , role of plant hormones, 165 

— and the life cycle of insects, 125 
— , selection of suitable biological, 

material for the study of, 219-222 
Dormancy-breaking in fungal 
spores, 110 

— in seeds, 166. 175 

— biochemical changes, 191 
Dormancy-induction, biochemical 

changes, 191 
Drying of bacteria 3 
Echinococciis granulosus, 101 



Ecdyson, 132 

Eggs of helminths, 97 

Electron transport system, in bac- 
terial spores, 82 

— , in seeds 197, 198 

Encysted larvae of helminths, 101 

Environmental factors, 144 

Enzyme pattern of dormant spores, 
75 

Escherichia coli, damage to cell wall 
by freezing, 18 

— , inactivation of dried cells by 
molecular oxygen, 27 

— , kinetics of death, 21 

— , sensitivity to lysis by lysozyme, 
19 

— , storage death of frozen cells, 17 

Ether, 110 

Ethyl acetate, 49 

— formate, 50 

— malonate, 49 

■ — oxamate, 55, 56 

— propionate, 50 

— pyruvate, 49, 61, 69 

— succinate, 50 
Ethylene, 8 

Far Red irradiation, 180, 181 

Flavin mononucleotide, 84 

Flavoprotein oxidase, 84 

Fluorescence quenchers, 26 

Fluoroacetic acid, 51 

Formic acid, 46 

Fraxinus, 169 

Free larvae of helminths, 99 

Freeze-drying, 20 

Freezing, 16, 17 

Fumaric acid, 46 

Fungistasis, 118 

Furfural treatment of fungal spores, 

110 
Fusarium fructigenum, 1 1 1 

— oxysporum, 1 1 8 

— so Ian i, 1 1 



SUBJECT INDEX 



229 



Germination, bacterial spores, 12, 

33, 65 
— , fungal spores. Ill 
— , seeds, 160, 175 
— , first step in, 69 
— , inhibition of, 167 
— , light effect on, 179, 183 
— , nutritional requirements for, 1 1 1 
— , prime event, 65 
— , salt inhibition, 68 
— , temperature, 33, 178 
— , trigger mechanism, 12, 64, 65, 68 
— , effect of versene on, 34 
Gibberellin, 105 

— , effect on dormancy in seeds, 166 
Glomerella cingidata, 1 1 1 
Glucose oxidation in bacterial 

spores, 79 
Glucose phosphate dehydrogenase, 

195 
Glutamic acid, 45, 46 
L-Glutamic acid diethyl ester, 49 
Glutamine, 45 
Glutathione, 23 
Glycerol, 19 
Glycine, 45 
Glycolic acid, 46 
Glyoxylic acid, 46 
— , shunt, 46,47, 51, 54, 58 
Gongylonema longispicula, 105 
Growth promoting substances in 

woody plants, 206, 207 
Growth inhibiting substances in 

woody plants, 206, 207 
Haemonchiis contort us, 100 
Heat sensitization of bacterial 

spores, 56, 57 
Heat shock of bacterial spores, 13, 

33, 64 
Helminthosporium sativum, 1 1 1 
— teres, 116 
Heterodera schachtii, 98 
Hexetedine, 80 



Hexokinase, 79 

Hibernation, 1, 104 

Histidine, 45 

Histiosoma, 125 

Histotropic phase of nematode lar- 
vae, 103 

[B-Hydroxybutyric acid, 46 

[3-Hydroxyglutamic acid, 45 

a-Hydroxyglutaric acid, 46 

8-Hydroxyquinoline, 82 

Hylemia antiqua, 156 

Hymenolepis nana, 105 

Hymenolepsis diminuta, 105 

Hypobiosis, in parasitic worms, 97 

— , due to hypobiosis of the host, 
104 

Hypobiotic phenomena in fungi, 107 

Hypometabolism. 1 

Hypoxanthine, 112 

Indolacetic acid, stimulation of ger- 
mination of uredospores, 117 

Indolepyruvic acid, 207 

Indoleethylacetate, 207 

3-Indolylacetic acid, 165 

Inosine, 79 

Iron, 48 

— , level in bacterial spores, 72 

Isocitric acid, 46 

Isoleucine, 88 

Isonicotinic acid, 44 

a-Ketoglutaric acid, 46 

a-Ketopimelic acid, 46 

Kinetin, 165, 166 

Lactic acid, 46 

Lactose, 23 

Lactuca sativa, 1 66 

Leucine, 88, 111 

Lipase, 194 

Lithium chloride, 68 

Locusta migratoria. 111, 144, 146 

Locustana pardaliua, 148 

Lyophylization, 2, 15, 20 

Lysine, 111 



230 



SUBJECT INDEX 



Magnesium, 48 

— , effect on germination of bacterial 

spores, 34, 77 
Magnesium chloride, 68 
Malic acid, 46 
Malonic acid, 48 
Manganese, 48 

— , level in bacterial spores, 72 
— , inhibition of ATPase, 82 
Maturation of fungal spores, 112, 

113 
Melanoplus differentia/is, 135, 151 
— bivittatus, 1 50 
Mercuric chloride, 68 
Mesotartaric acid, 46 
Metabolism in diapausing insects, 

138 
Metacercaria, 101 
a-Methylglucoside, 23 
DL-a-Methylglutamic acid, 46 
Methylthiourea, 23 
Microsclerotia, 1 14 
Mineral oil sprays, 205 
Miracidium, 99 
Monilia fructigena, 1 1 1 
Moniliforniis moniliformis, 105 
Mormoniella, 137 
Mycosphaerella pinodes, 1 1 6 
Naringenin, 207 
Naylor's medium, 20 
Nematodes, 99, 101 
Neoascaris vifulorum, 102 
Neurospora crassa, 1 1 
Nickel, inhibitor of germination of 

bacterial spores, 34 
— , level in bacterial spores, 72 
— , effect on inhibition of sporula- 

tion, 48 
Nicotinic acid, 44, 114 
Nomadacris septemfasciata, 1 50 
Nucleoside ribosidase, 79 
Nucleoside phosphorylase, 79 
Octyl alcohol, 12 



— , effect on bacterial spores and 
vegetative cells, 55 

— , action on trigger reaction, 69 

Osmobiosis, 15 

Outgrowth in bacterial spores, 33 

Oxalacetic acid, 46 

Oxalic acid, 46 

Oxygen, demand in bacterial cul- 
tures during growth and sporula- 
tion, 40 

— , lethal effect on dried bacteria, 
22, 25 

— , uptake in dormant spores, 74 

Papaver rhoeas, 108 

Panonychits ulmi, 122, 126 

Pareupiepocnemis syriacus, 144 

Peronospora schleideni, 109 

Peroxidase in dry seeds ,195 

Phenolase in dry seeds, 195 

Phosphoglucomutase, 79 

Phosphohexokinase, 79 

Phosphoribomutase, 79 

Photoperiod, in diapause, 121, 122, 
126, 156 

— , resting stage of buds, 205, 206 

Phycomyces blakesleeamis. 1 1 2 

Phytase, 194 

Phytin, 197 

Phytophtora cactorum, 113 

a-Picolinic acid, 43 

— , as inhibitor of bacterial sporula- 
tion, 44 

a-Pimelic acid, 46, 60 

Pipecolic acid, 46 

Plasmodiophora brassicae, 109 

Plasmopara viticola, 1 1 6 

Plerocercoid, 101 

Polypedilum vanderplanki, 121 

Potassium chloride, 68 

Proline, 111 

Propionic acid, 46 

Protective colloids, 18, 26 

Proteolytic enzymes in bacterial 



SUBJECT INDEX 



231 



spores, 36 
Pseudolifelessriess, 1 
Piiccinia gvaminis, 109. 113 
Pyrophosphatase. 75 
Pyruvate hypothesis, 80 
Pyruvic acid, 42, 46, 197 
Quiescence, 1 
Quinol, 197 
QuinoHnic acid, 44 
Red-Far Red reaction. 180. 181 
Rehydration of bacteria, 3 
Reseda odorata, 108 
Respiration, in diapausing insects, 

138 
— , in dormant spores, 74 
— , in seeds, 5, 186, 187, 188 
Resting fungal spores, 108, 110 
Resting of nematode larvae in the 

unborn foetus, 102 
Rhabditis coarctafa, 100 
Rhabditis maupasi, 101 
Rhizoctonia solani, 116 
Riboflavin phosphate, 26 
Ribokinase, 79 
Ribosidase, 36 
Saturnia pavonia, 143 
Schistosoma mansoni, 105 
Schistosoma haematobium, 106 
Sclerotia, production of, 113, 114, 

116, 118 
Sclerotinia sclerotiorum, 114 
Sclerotium rolfsii, 114, 118 
Scopoletin, 208 
Serine, 88 
Significance of the enzyme activities 

of dormant spores, 76 
Schistocerca gregaria, 144 
Sodium bisulfite, 53 
Sodium chloride, 68 
Sodium iodide, 23, 27 
Sodium nitrate, 68 
Spirocerca htpi, 102 
Storage of dried cells, 4 



Stratification, 177 

Succinic acid, effect on inhibition of 

sporulation, 45, 46 
— , intermediate in synthesis of spore 

material, 54, 55, 58 
Succinic cytochrome reductase, 83 
Tetranychiis iirticae, 128 
Thioacetamide, 23 
Thiocyanate, 8 
Thiamine, 128 
Thiourea, breaking of dormancy in 

buds, 8 
— . germination stimulator of seeds, 

169, 179, 192 
— , metabolism of fats in seeds, 193, 
— , oxidative system in seeds, 197 
— , protective eff'ect on bacteria, 27 
Threonine, 88 
Tilletia controversa, 109 
Torulopsis sanguinea, 1 1 2 
Tmethis pidchipeunis asiaticus, 144, 

146 
Toxocara canis, 102 
Trematodes, 99, 101 
Tricarboxylic acid, cycle, in seeds, 

195, 197 
— , in spores, 46, 51, 80 
Triethylcitrate, 49 
Trigger mechanism, in buds, 8 
— , in cysts, 9 

— , in hatching of insect eggs, 10 
— , in seeds, 6 
Trigonella arabica, 1 70 
Trimethyl thiourea, 23 
Trogoderma granaria, 141 
Tropaeohim majus, 108 
Trypticase, 17 
Urethan, 23 
Urocystis tritici, 1 1 1 
Uromyces phaseoli, 1 1 3 
Ustilago striiformis, 109, 110 
Valine, 88 
Versene, 34, 48, 82 



232 SUBJECT INDEX 

Vitis ber/andieri, 206 Winter eggs in helminths, 98 

Vitis rupestris, 206 Zinc, 48 

Vitis vinifera, 206 Z-factor in fungal spore germination 

Water content of bacterial spores, 71 112